The effect of lanthanum (III) on the activity of xylanase by Penicillium and its influence on brightness in the paper pulp bleaching

Abstract

Xylanase is widely used in pulp and paper bleaching. In this study, the effects of rare earth ions (La³⁺, Ce³⁺, Er³⁺ and Gd³⁺) on the activity of xylanase produced by Penicillium are investigated and the application of a xylanase solution containing La³⁺ in paper bleaching is presented. Our results indicate that the bleaching effect of the enzyme solution containing La³⁺ was markedly better when the concentration of La³⁺ was 10⁻⁸ g/L after 4 days of incubation. The mechanism of lanthanum on the improvement of xylanase activity was revealed through electrical conductivity, atomic absorption spectrometer, infrared spectroscopy and fluorescence microscopy analyses. The PCR result clearly demonstrates that a low concentration of La³⁺ led to the transversions of three base pair of gene sequences. Our experiment also reveals that the La³⁺ may have been involved in the cellular metabolic processes of Penicillium and intervened in the base pairing and DNA replication. This research may provide new insights into the improvement of enzymatic activity by lanthanum (III) and its application in paper pulp bleaching.

Introduction

In recent years, rare earth elements (REEs) have been widely studied owing to their applications in agriculture, animal husbandry, industrial and biomedical fields (Zhang et al. 2015; Wang et al. 2014; Chen et al. 2012; Zhang et al. 2009; Vijayaraghavan et al. 2011; Wang et al. 2017). Lanthanum (La) is one of the important REEs widely present in the earth’s crust. Many studies have shown that the effects of La on plants depend on its concentration. A few REEs at low concentrations in living organisms can promote enzymatic activity; conversely, at high concentrations can inhibit the activity of enzymes (Furst 1987). Lots of studies have attempted to reveal the effects of REEs and the corresponding mechanisms. For instance, the bioaccumulation of La and its effects on the growth and mitotic index of soybeans have been investigated (de Oliveira et al. 2015). Low levels of La(III) promoted plant photosynthesis and growth, whereas high levels of La(III) inhibited plant photosynthesis and growth owing to destruction of the chloroplast ultrastructure (Hu et al. 2016). A low concentration of La(III) made the molecular structure of calmodulin more compact and orderly, but a high concentration of La(III) interacted with cytoplasmic calmodulin and thus made its molecular structure looser and more disordered (Wang et al. 2016). Pretreatment with 20 mg/L La(III) alleviated the inhibitory effect of ultraviolet-B (280–320 nm) radiation on the assimilation of nitrogen by soybean seedlings (Huang et al. 2013). It was also found that a low concentration of La could protect roots against salt stress and limit the wilting of plants (Shan and Zhao 2014). Studies have also shown that La can improve the Cd tolerance of Zea mays seedlings by regulating the metabolism of ascorbate and glutathione (Dai et al. 2017). Ce is another important light REEs, and Er and Gd are two abundant heavy REEs in the earth’s crust. These elements have been reported to be involved in biological effect. For instance, the research of Xia et al. (2013) showed that Ce³⁺ enhanced the activities of antioxidative enzymes in adventitious shoots and Chen et al. (2012) showed that Ce³⁺ affected the antibacterial mechanism of Escherichia coli cells. In addition, Gd affected the antioxidant enzymes in goldfish livers (Chen et al. 2000) and Er³⁺ may have negative effect on bone metabolism (Zhang et al. 2011). Therefore, in this study, the effects of four abundant REEs (La³⁺, Ce³⁺, Er³⁺ and Gd³⁺) on the activity of xylanase have been analyzed.

Xylanase can be effectively used to hydrolyze xylan (Knob and Carmona 2010; Collins et al. 2005) to substituted xylooligosaccharides with different chain lengths (Chi et al. 2015; Walia et al. 2017). Xylanase are employed, for example, in fruit maturation, seed germination, fungal parasitization, kraft pulp bleaching, food baking and animal feed preparation (José et al. 1999). In particular, in the paper industry, xylanase can reduce the amounts of chemical bleaching agents used, increase brightness, and greatly reduce environmental pollution, and importantly, they represent an economical and cheap technology (José et al. 1999). Xylanase can be derived from a variety of microorganisms. The focus of our experiment, in contrast to bacteria, plant and animal cell, is on the microbial xylanase because of its ease of obtainability. In recent years, xylanase production and the improvement of xylanase activity have attracted close attention. However, most of the research on improvement of xylanase production has focused mainly on fungal growth and the optimization of cultivation conditions. The improvement of xylanase activity by metal (REEs) ions has seldom been reported in global publications. Commercial applications require cheaper xylanase, higher levels of enzyme expression and the efficient secretion of xylanase to make the process economically viable. Therefore, improvement of the activity of xylanase may be an effective means of achieving this.

The aim of this study, in view of the promotion of enzymatic activity by La(III) at low concentrations, is to determine whether and how REEs regulate the activity of xylanase produced by Penicillium strains. We have attempted to interpret the effect of La(III) on the production of xylanase by Penicillium and the corresponding mechanism. Our results provide new insights on the possible applications of La(III) in paper production.

Materials and methods

Strains and experimental materials

Strains were separated from soil samples obtained from the Inner Mongolia University of Science and Technology (IMUST) and identified as Penicillium strains by the Research Institute of Microbiology of the Chinese Academy of Sciences. The pulp used in our experiments was a mixture of synthetic tree pulp and poplar pulp. The testing of materials include wheat bran, yeast extract, MgSO₄, NaCl, KH₂PO₄, rare earth chlorides, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 8-anilino-1-naphthalenesulfonic acid (ANS), phosphate-buffered saline (PBS) buffer solution, maleic acid and 0.05 mol/L Tris–HCl (pH 7.4). DNS is made up of potassium sodium tartrate (182.09 g), 3,5-dinitrosalicylic acid (6.39 g), NaOH (21.09 g), phenol (5.09 g) and Na₂SO₃ (5.09 g). All these were all purchased from Tianwei Biotech Co., Ltd (Beijing). All of the other chemical reagents were of an analytical grade. The rare earth chlorides used hereby were LaCl₃, CeCl₃, ErCl₃ and GdCl₃. These were dissolved in deionized water to prepare solutions of different concentrations (10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸ and 10⁻⁹ g/L).

Enzyme assay

Solutions of rare earth chlorides were added to a basic medium. After 24 h of incubation, the culture solution (2 mL) was transferred to a centrifuge tube every day. It was centrifuged at 5000 r/min for 10 min. The supernatant was the crude enzyme solution. Then, 1.9 mL of xylan [1% (w/v), pH 4.8] solution and 0.1 mL of the crude enzyme solution were mixed in a test tube and placed in a water bath at 50 °C for 30 min. And adding 3 mL of DNS solution after taking out immediately. The test tube was placed in a boiling water bath to maintain the temperature and develop color for 5 min. After the temperature of solution cooled down to 25 °C, 10 mL of distilled water was added and the OD value at 620 nm was measured using a spectrophotometer. A buffer solution containing 1% xylan was selected as the substrate for the enzymatic reaction. One unit of xylanase activity was defined as 1 mL of the crude enzyme solution, which was used to decompose xylan per minute to produce 1 μg of xylose at 50 °C, 1% xylan substrate solution at pH 4.8.

Analysis of the effects of REEs on the production of xylanase by Penicillium

The solutions of rare earth chlorides were added in different proportions to a basic medium. The rare earth chlorides (LaCl₃, CeCl₃, ErCl₃ and GdCl₃) were added until the final concentration of the corresponding REE was 0, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸ and 10⁻⁹ g/L, respectively. Afterward, the resulting solutions were sterilized and injected into Penicillium. The cultures were then shaken at a constant temperature (30 °C, 170 rpm) for about 48 h. The activity of xylanase was measured every 24 h. The lanthanum solution (10⁻⁸ g/L) was selected because the activity of xylanase produced by Penicillium was greater. Then, three groups of parallel samples were prepared in the above manner. LaCl₃ was added to the liquid medium until the final concentration of La was 10⁻⁸ g/L. The Penicillium strain was cultured for 24 h at a constant temperature with shaking (30 °C, 170 rpm). The activity of xylanase was then measured over 5 days.

The xylanase solution to which LaCl₃ had been added was reacted with xylan at 30, 40, 50, 60, 70, and 80 °C for 30 min, respectively. Three groups of parallel samples were prepared according to the above method. The activity of xylanase was assayed after the mixtures had reacted for 30 min. LaCl₃ was added to the xylanase solution, and the temperature was maintained for 1 h at 30, 40, 50, 60, 70 and 80 °C, respectively. After the solution had cooled, the enzymatic activity was also measured. The highest enzymatic activity was defined as a relative enzymatic activity of 100%. Three parallel experiments were carried out at pH values of 3.6, 4.8, 5.4, 6.0, 6.6, 7.2 and 7.8, respectively. A buffer solution containing 1% xylan was selected as the substrate for the enzymatic reaction. The enzymatic activity was subsequently measured. The highest enzymatic activity was defined as a relative enzymatic activity (at a pH of 4.8) of 100%. The enzyme solution was diluted with a buffer solution with a pH of 3.6, 4.2, 4.8, 5.4, 6.0, 6.6, 7.2 and 7.8, respectively. Then, the diluted enzyme solution was incubated at 50 °C for 2 h, and the enzymatic activity was measured. The relative enzymatic activity at the maximum absorbance was defined as 100%.

Determination of electrical conductivity of Penicillium fermentation medium

The LaCI₃ solution was mixed with the enzyme solution to ensure that the concentration of La³⁺ in the solution was 0 g/L, 10⁻¹ g/L and 10⁻⁸ g/L, respectively. The activated strain was added in a volume fraction of 3% after being sterilized at 121 °C for 20 min. The cultures were shaken at a constant temperature (30 °C, 170 rpm) for about 24 h. The electrical conductivity was measured at room temperature per day.

Determination of Ca²⁺ in cell culture

Penicillium strains with concentrations of La³⁺ of 0, 10⁻¹ and 10⁻⁸ g/L, respectively, were cultured under optimum conditions. Samples were prepared by filtering and drying and ground into a powder using a mortar. A 0.1-g dry sample was accurately weighed and dissolved in a ratio of 1:4 in nitric acid, and the solution was then diluted to 100 mL. Three groups of parallel samples were prepared in this manner. The contents of calcium were measured using an atomic absorption spectrometer (AAS).

Analysis of dry samples by infrared spectroscopy

The dry product was characterized by spectroscopic analysis using Fourier transform infrared spectroscopy.

Observation of protoplast suspensions by fluorescence microscopy

Penicillium strains were cultured with La³⁺ in concentrations of 0, 10⁻¹ and 10⁻⁸ g/L, respectively, under optimum conditions. The resulting cell suspension was collected in a test tube by suction filtration. Then, an appropriate mixture of lysozyme and snailase with a concentration of 10 mg/mL was added, and the cell walls were hydrolyzed for 30 min in a 35 °C water bath. The cell suspension was centrifuged (1000 rpm, 10 min) after enzymatic hydrolysis, and the supernatant was collected and centrifuged (12,000 rpm, 10 min) again. The supernatant was removed as well as the remaining precipitate comprised protoplasts, which were then fully suspended in a solution of stabilized minimal medium. The ANS was diluted with a solution of PBS buffer (pH 7.2) to a concentration of 5.0 × 10⁻³ mol/L. Prior to the experiment, the ANS solution was further diluted to a concentration of 1.0 × 10⁻⁴ mol/L. An aliquot of 0.5 mL of the protoplast suspension from each of the three experimental groups was transferred to a 10-mL centrifuge tube, to which 2 mL of the ANS solution was added and mixed well. A small amount of prepared protoplast suspension was obtained and placed on the sample plate of a fluorescence microscope for observation. In this experiment, fluorescence microscopy was also performed on the unbroken cells.

Effect of La³⁺ on the enzyme-producing gene of Penicillium

Chromosomal DNA was extracted from the Penicillium strains using a TIANamp bacterial DNA Kit in the light of the instruction book. The isolated DNA was separated by electrophoresis on a 1.2% agarose gel. For primer design, a BLAST search of the GenBank database (http://www.ncbi.nlm.nih.gov/genbank) was conducted to find DNA sequences with significant homology to those xylanase gene from Penicillium (Zhang et al. 2009). The following primers were used: 5′-ACGGAGGCTGGGTAAATTCA-3′ as the plus strand and 5′-TAGTATCAAACCGCCAGCCT-3′ as the minus strand. Amplification cycling was performed as follows: 94 °C for 5 min, 94 °C for 30 s, 53 °C for 30 s, 72 °C for 60 s for 30 cycles, and 72 °C for 10 min. The polymerase chain reaction (PCR) amplification products were cloned one by one and subsequently purified using a TIANgel Midi Purification Kit (Tianwei Biotech Co., Ltd, Beijing). Fully automated bidirectional sequencing was performed using the same primers, which were acquired from the Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Zhang et al. 2009).

Influence on brightness in the paper pulp bleaching

A 2.0-g sample of homemade pulp was accurately weighed, and 100 mL of water was added and blended with it. The pulp was then treated with 2% hydrogen peroxide and 1% NaOH and was also bleached with the crude xylanase solution (0.4, 0.6, 0.8, 1.0 and 1.2 mL, respectively). The brightness of the pulp was measured for comparison with the values recorded for the pulp treated with the enzyme solution without rare earth ions. The brightness of the pulp was measured using a WSB-1 whiteness meter (Simulation D65 lighting) according to the Chinese National Standard GB/T 7933-87.

Results and discussion

Effect of rare earth ions on the production of xylanase by Penicillium

As shown in Fig. 1, changes occurred in the activity of xylanase with the addition of La³⁺, Ce³⁺, Er³⁺ and Gd³⁺. In contrast to Er³⁺ and Gd³⁺, the influences of La³⁺ and Ce³⁺ on the activity of this enzyme were more evident. More specifically, the enzymatic activity increased by 36.6% in 4 days when the La³⁺ concentration was 10⁻⁸ g/L and by 41.5% in 3 days when the Ce³⁺ concentration was 10⁻³ g/L. Study has shown (Yang et al. 2012) that the structure of proteins on the plasma membrane of horseradish that undergoes changes in response to Ce³⁺ may change the permeability of the cell membrane, which increases or decreases the exchange of substances between the interior and the exterior of the cell. Thus, it is possible that different amounts of rare earth ions may also affect the permeability of the cell membrane.

Fig. 1.

Effect of rare earth on xylanase activity a Er³⁺, b Gd³⁺, c La³⁺, and d Ce³⁺

Effect of incubation time with La³⁺ on activity of xylanase produced by Penicillium

La³⁺ and Ce³⁺ both greatly increased the activity of xylanase produced by Penicillium. Because the respective Ce³⁺ concentration was higher and the high cost of rare earth was taken into consideration, only La³⁺ was selected for the subsequent experiments. From Fig. 2a, it can be seen that different incubation times with rare earth ions had greater effects on the activity of xylanase produced by Penicillium. In particular, when the concentration of La³⁺ was 10⁻⁸ g/L the activity was greatly enhanced. The xylanase activity increased by 9.6%, 16.1%, 24.7%, 54.8% and 23.7% after culture times of 1, 2, 3, 4 and 5 days, respectively. Therefore, it was proved that the most suitable culture time was 4 days.

Fig. 2.

Effects of La³⁺ on xylanase. a Effect of the culturing time on relatively enzymatic activity; b effect of temperature on xylanase activity; c thermal stability of xylanase; d effect of pH on xylanase activity; e pH stability of xylanase

Effects of La³⁺ on xylanase

As can be seen from Fig. 2b, in the temperature range of 40–60 °C, the enzymatic activity conspicuously increased whether or not La³⁺ was added. In particular, at 50 °C the activity was the greatest. It is clearly indicated in Fig. 2c that the xylanase activity was almost unchanged in the range of 30–50 °C in the absence of La³⁺. The activity of this enzyme remained above 99.0%. Nevertheless, as the temperature was gradually increased, the activity clearly decreased. In comparison with the above results, the activity was also stable at above 96% in the presence of La³⁺ between 30 and 60 °C. Therefore, the maximum temperature at which the enzymatic activity was stable increased by 10 °C in comparison with the counterpart without La³⁺. Figure 2d shows that the enzymatic activity was the highest in the absence of La³⁺ at a pH level of 4.8. Furthermore, the activity remained over 95% while the pH value was in the range of 4.8–6.6. However, the enzymatic activity reached its maximum when the solution contained La³⁺ and the pH was about 5.4. Consequently, the activity remained high from a pH of 4.8 to a pH of 6.6, and its average value increased by 5.7%. Our results (Fig. 2e) have shown that the rare earth ions had little effect on the pH stability of xylanase. Whether La³⁺ was added or not, the enzymatic activity was high (> 90%) when the pH was approximately 4.8–6.0. In general, we have analyzed the essence of the interaction between REEs and xylanase from two different aspects. The first is the effect of REEs on the enzyme itself, and the second is the influence of rare earth on bacteria. Thus, we conclude that La³⁺ has weak effect on the enzyme itself according to the above experimental results.

Influence of La³⁺ on conductivity of Penicillium fermentation medium

In general, the opening or closing of ion channels in the cytoplasmic membrane will change with the physiological conditions in the cell, and the permeability of the cell membrane will also be changed by environmental conditions. This will affect the movement of ions into or out of the plasma membrane and lead to differences in ion concentration. When pure Penicillium is cultured with different concentrations of La³⁺, the La³⁺ ions may bind to phospholipid molecules on cell membrane proteins or in the phospholipid layers to change the structure of these phospholipids. Then, the cell structure may also change. When the structure of the cell membrane is found to change, its permeability may increase and soluble substances may permeate through the cell membrane. Consequently, the conductivity will change. The relevant experimental data are shown in Fig. 3a. It can clearly be seen that different conductivity values were measured at the same time after Penicillium had been cultured with different concentrations of La³⁺. Our experimental results show that when the concentration of La³⁺ was 10⁻¹ g/L, the conductivity was significantly higher than that in the absence of La³⁺ on the second day of culture. From a comparison of the results for the three concentrations, the conductivity at a high rare earth ion concentration level was significantly greater than that at a low rare earth ion concentration. When the concentration level of La³⁺ was 10⁻⁸ g/L, the conductivity was slightly higher than that in the absence of La³⁺, and the change in conductivity was not obvious on the second day of culture. The conductivity increased with an increase in culture time. Our experimental results show that the conductivity of the culture solution with La³⁺ was significantly greater than that of the solution without La³⁺. This can be explained by the fact that the substances inside the cell membrane underwent osmosis, whereas the permeability of the cell membrane slowly increased and was greater at a high rare earth ion concentration level.

Fig. 3.

The analysis of conductivity, AAS, IR spectra, and fluorescent microscopy images. a The conductivity of three kinds of bacteria liquid; b content of Ca²⁺ in Penicillium; c IR spectra of cell wall of Penicillium; d fluorescent microscopy images of protoplast in Penicillium treated with 0 g/L (A), 10⁻¹ g/L (B), 10⁻⁸ g/L (C), La³⁺ in the present of ANS, respectively

Determination of Ca²⁺ in cell culture

The change of Ca²⁺ in the cell culture is further evidence of the production of xylanase by Penicillium. So, the contents of calcium ions in the cells were quantitatively determined using an AAS. The results of the measurements are shown in Fig. 3b. It can be observed from the experimental results that the concentrations of the Ca²⁺ were different when the strains had been cultured in the presence of different La³⁺ concentrations. As the concentration of La³⁺ (from 10⁻¹ to 10⁻⁸ g/L) in the solution decreased, that of calcium ions increased. When the concentration of La³⁺ was 10⁻⁸ g/L, the calcium content was the greatest; that is to say, when the enzymatic activity reached its maximum, the content of Ca²⁺ was the highest. Because the ionic radius of rare earth and Ca²⁺ ions are similar, rare earth ions often act as an antagonist of, or substitute for, calcium ions in organisms, which interferes with the normal physiological functions of calcium. There are two distinct mechanisms of Ca²⁺ transport in bacteria. In one pathway, the concentration of Ca²⁺ in the cytosol is increased by calmodulin, and the activation of calmodulin leads to the initial cell effect. The other pathway involves C-kinase. An increase in the concentration of calcium ions is responsible for a rise in the diacylglycerol content of the plasma and a sustained phase of cell response (Rasmussen and Barrett 1984). A low concentration of La³⁺ replaced the Ca²⁺ bound to calmodulin, which increased the intracellular Ca²⁺ concentration and the production of enzymes by the above two distinct mechanisms. An increase in the concentration of La³⁺ caused more Ca²⁺ ions to be lost. As a result, the ability of the strain to produce enzymes decreased. In short, the change in the concentration of Ca²⁺ had a vital influence on the production of xylanase by Penicillium because the changes in the Ca²⁺ content and the enzymatic activity were basically identical. This phenomenon was mainly caused by the addition of rare earth ions.

Infrared spectroscopic analysis of cell samples

The purpose of this section is to provide proof of a formation of a new compound in cell plasma due to the involvement of La³⁺. The results of our infrared spectroscopic analysis are shown in Fig. 3c. The infrared spectral frequencies of the respective functional groups are listed in Table 1, which shows the vibrations of the groups in the external cell wall of Penicillium. Shifts occurred in the absorption peaks at 3325/cm and 3406/cm, which represent the stretching vibrations of the N–H bonds in proteins and the O–H bonds in water in carbohydrates, respectively. A change occurred in the absorption peak at 1648/cm, which is due to –CH₂ and –CH₃ groups in lipids and P=O bonds in phosphate compounds. Figure 3c shows that there was a manifest shift in the absorption peak at 1050.71/cm. This peak is due to the stretching of the C–O–C groups in polysaccharides (Avelino et al. 2018). The reason for this may be that a low concentration of La³⁺ promoted cell growth and affected the cross-linked structures of polysaccharides. The peak below 600/cm in the spectrum is due to the vibrational absorptions of the M–O and O–M–O groups (where M is a metal ion) (Rasmussen and Barrett 1984). The reason for the shifts in the peaks may be that La³⁺ ions replaced the calcium or magnesium ions in the cell plasma and formed new chelating ligands with oxygen. This indicates that La³⁺ had an effect on groups in the external cell wall of Penicillium. La³⁺ ions in high concentrations may form coordinate bonds with groups on the cell wall or cleave linkages, which can destroy the structure of the cell wall and inhibit the growth of the cell. La³⁺ ions in low concentrations only changed the cross-linked structures of polysaccharides and did not cleave bond in linkages. However, they increased the permeability of the cell wall, which is beneficial for the growth and reproduction of cells and can increase enzymatic activity.

Table 1.

Significant IR absorption frequencies of cell wall (cm⁻¹)

Constituents of La3+ (g/L)	νO–H, νN–H	νCH2, νCH3, νP=O	νC–O–C	νM–O
0	3325.84306	1648.68419	1050.71583	535.32405
10−1	3389.9111	1657.22659	1043.59716	536.74779
10−8	3405.57218	1652.95539	1029.35982	529.62912

Fluorescence microscopy

Fluorescence microscopy analysis further confirms the interaction La³⁺ with the structures of proteins on the plasma membrane of Penicillium. Firstly, Penicillium was cultured in La³⁺ solutions of three different concentrations, and the fluorescence intensity of Penicillium was measured under different conditions. The effects of the fluorescence intensity on the proteins of the cell membrane and cell wall at different concentrations of La³⁺ are shown in Fig. 3d. After the protoplasts of Penicillium and ANS had reacted for 1 min, a membrane protein–ANS complex system was produced by the combination of ANS with nonpolar groups of membrane proteins of Penicillium. As shown in Fig. 3d-C, this compound emitted fluorescence at a La³⁺ concentration of 10⁻⁸ g/L. This indicates that La³⁺ may have interacted with the membrane proteins of Penicillium, which can change their structure or promote their synthesis. A strong complex of ANS with the membrane proteins of Penicillium was formed, so that brighter fluorescence occurred. When the concentration of La³⁺ was 10⁻¹ g/L, hardly any fluorescence could be observed (Fig. 3d-B). This occurred because a high concentration of La³⁺ can destroy the structure, as well as prevent the synthesis of membrane proteins. The reason why the fluorescence intensity was weaker may be that the combination of ANS with the membrane proteins of Penicillium was limited.

Cloning of xylanase genes

The concentrations of genomic DNA cultured at La³⁺ concentrations of 0, 10⁻¹ g/L and 10⁻⁸ g/L, as the result of the determination of the concentrations of three extracted DNA samples, were found to be 3600, 3100 and 4700 ng/μL, respectively. The three corresponding PCR products are shown in Fig. 4. After they were recovered, sequencing was carried out. During this process, the amplified product from the strain cultured at a La³⁺ concentration of 10⁻¹ g/L was rapidly degraded and could not be sequenced. The comparative results for the other two sequences are shown in Fig. 4. The entire sequences of the xylanase gene were reconstructed using DNAMAN software. After a comparison using BLAST, it was concluded that 99% of the gene sequences were identical in both cases with 1000 base pair matches. Exactly, three base pair transversions were found in the xylanase gene produced by the Penicillium that was treated with La³⁺. However, no transition was occurred in the xylanase gene sequence. A was mutated to G, and G was mutated to A. This interesting result of gene transversions could be due to the fact that the xylanase activity was greatly enhanced with La³⁺ (10⁻⁸ g/L). More specifically, a low concentration of La³⁺ led to three transversions in the gene sequences, whereas a high concentration of La³⁺ had a great influence on DNA and caused it to degrade in a short period of time.

Fig. 4.

Amplification of xylanase genes: M, marker; A, 0 g/L (La³⁺); B, 10⁻¹ g/L (La³⁺); C, 10⁻⁸ g/L (La³⁺). Sequence comparison of the xylanase genes: top strand, 0 g/L (La³⁺); Bottom strand, 10⁻⁸ g/L (La³⁺)

Influence on brightness in the paper pulp bleaching

Xylanase is widely applied in pulp and paper bleaching. The experimental results showed that the brightness of the unbleached original pulp was 22.2. The brightness of paper pulp preprocessed with H₂O₂ and NaOH was 32.8. When 0.6-mL enzyme solution containing La³⁺ was added, its brightness was 52.8. The brightness was, thus, 30.6 higher than that of the original pulp. The bleaching effect of the 0.6-mL enzyme solution containing La³⁺ was, thus, obviously better (Fig. 5). This could be accounted for by the fact that the addition of lanthanum ions increased the enzymatic activity. Obviously, the addition of La³⁺ can improve the enzymatic activity and reduce the dosage of the xylanase solution in pulp bleaching (Fig. 6).

Fig. 5.

Effect of paper bleaching (A, denoted no La³⁺, B, stand for with La³⁺)

Fig. 6.

The brightness of the pulp under different conditions [A, original pulp paper; B, bleaching by H₂O₂ (2%); C, bleaching by xylanase (0.6 mL); D, bleaching by xylanase adding La³⁺ (0.6 mL)]

Conclusions

Our experimental results demonstrate that the activity of xylanase was changed by adding four kinds of rare earth ions (La³⁺, Ce³⁺, Er³⁺ and Gd³⁺). In contrast to Er³⁺ and Gd³⁺, the influences of La³⁺ and Ce³⁺ on the activity of this enzyme were more evident. In particular, when the concentration of La³⁺ was 10⁻⁸ g/L the activity was greatly enhanced. The enzymatic activity was increased by a change in the structure of the enzyme at a low concentration of lanthanum ions. The addition of lanthanum ions increased the enzymatic activity. When a 0.6 mL enzyme solution containing La³⁺ was added to pulp, its brightness was 30.6 greater than that of the original pulp. The amount of enzyme solution containing rare earth ions was less, but the outcome was improved. The experimental results showed that the conductivity of the culture solution with La³⁺ was significantly higher than that of the corresponding solution without La³⁺. A low concentration of La³⁺ replaced Ca²⁺ bound to calmodulin, which increased the intracellular Ca²⁺ concentration and the production of enzymes by the above two distinct mechanisms. A low concentration of La³⁺ changed only the cross-linked structures of the polysaccharides and did not cleave bonds in linkages. However, it increased the permeability of the cell wall, which is beneficial for the growth and reproduction of cells. A high concentration of La³⁺ can destroy the structure as well as prevent the synthesis of membrane proteins. A low concentration of La³⁺ produced some changes on the gene sequences, whereas a high concentration of La³⁺ had a great effect on DNA and caused it to degrade in a short time. Experimental results show that the rare earth ion La³⁺ may have been involved in the cellular metabolic processes of Penicillium and intervened in base pairing and DNA replication. Our research may provide new insights into the improvement of enzymatic activity by lanthanum (III) and its application in paper pulp bleaching.

Compliance with ethical standards

Conflict of interest

Authors declare that they do not have any conflict of interest.