A pH-stable alkaline pectate lyase produced by the newly identified strain Bacillus altitudinis CAS-WZS-08

2.1 Media and reagents

Isolation medium: pectin (galacturonic acid ≥ 74.0%, Shanghai Macklin Biochemical Co., Ltd.) 2 g/L, (NH₄)₂SO₄ 10 g/L, NaCl 2 g/L, KH₂PO₄ 0.3 g/L, K₂HPO₄ 1.0 g/L, MgSO₄ 0.3 g/L, FeSO₄ 0.1 g/L, agar 15 g/L, pH 7.0, sterilized at 115 °C for 30 min. Primary fermentation medium: pectin 2 g/L, (NH₄)₂SO₄ 10 g/L, NaCl 2 g/L, KH₂PO₄ 0.3 g/L, K₂HPO₄ 1.0 g/L, MgSO₄ 0.3 g/L, FeSO₄ 0.1 g/L, pH 7.0, sterilized at 115 °C for 30 min. Regents were purchased from Sinopharm Chemical Reagent Co., Ltd. Shanghai, China.

2.2 Isolation and identification of strain

2.3 Determination the enzyme activity

Enzyme activity was measured using the 3, 5-dinitrosalicylic acid (DNS) method. To obtain the crude enzyme, 1.0 mL of freshly grown culture was centrifuged at 10, 000 rpm for 10 min. The substrate was prepared by mixing 0.1% (w/v) pectin in Glycine-NaOH buffer (50 mmol/L). The control experiment was prepared using a deactivated enzyme (100 °C, 5 min) instead of the normal pectate lyase. The supernatant (10 μl) was mixed with 500 μl substrate and incubated at 60 °C for 30 min. Adding the DNS solution (500 μl), then boiled the reaction mixture 5 min for detecting the absorbance at 540 nm. Enzyme activity (U/mL) is defined as the amount of enzyme required to catalyze the substrate to form 1 μmol of galacturonic acid per minute under the given conditions.

2.4 Effects of fermentation parameter

To obtain a higher enzyme activity, fermentation parameters, including pectin concentration, nitrogen types, nitrogen concentration, the initial pH of the media, inoculum size, and culture temperature, were determined step by step using single-factor analysis at one time (El-Ghomary, et al., 2021). The pectin concentration was designed for 2, 4, 6, 8, and 10 g/L. Nitrogen types, including urea, NH₄Cl, (NH₄)₂SO₄, yeast extract, tryptone, and beef powder, were measured at 10 g/L. In nitrogen concentration, seven concentrations were designed, including 4, 8, 12, 16, 20, and 24 g/L. Initial pH of the media was set to 5, 6, 7, 8, 9, and 10. Moreover, the inoculum size (v/v), including 1%, 2%, 3%, 4%, 5%, 6%, and 7%, were performed. Experiments were performed in triplicate (n = 3). Statistical analysis was carried out using analysis of variance in SAS studio. The p-values were compared to the significance level of 5% and used to determine the significance.

2.5 Characterization of enzyme property

Regarding the enzyme properties, the optimum temperature and stability in temperature, the optimum reaction pH and pH stability, the effects of metal ions on enzyme activity, and effects of buffer concentration were organized.

The optimum temperature was determined from 45 to 60 °C (tested in interval of 5 °C) in Glycine-NaOH (50 mmol/L, pH 9.0). To determine the temperature stability, the crude enzyme was preincubated at 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C, determining the remaining enzyme activity. The optimum pH was determined at 60 °C for 30 min in different buffers (50 mmol/L) with pH from 3.0 to 10.5, including Na₂HPO₄-Citric acid, KH₂PO₄-NaOH, Tris-HCl, and Glycine-NaOH (Jadhav and Pathak, 2019). The pH stability was determined by measuring the relative enzyme activity (Jalil and Ibrahim, 2021) after the enzyme incubation at 4 °C at different pH buffer from 3.0 to 10.0. The enzyme activity without preincubation was defined as 100%. Metal ion influences were calculated through 1 mM K⁺, Ca²⁺, Mg²⁺, Zn²⁺, Mn²⁺, Cu²⁺, Ni²⁺, Fe²⁺, Fe³⁺, Ba²⁺, and Co²⁺ in the reaction mixture. The buffer concentrations were set as 0, 50, 100, and 200 mmol/L to determine the influence of buffer concentrations.

2.6 Purification of pectate lyase

To obtain the purified enzyme, several purification methods were evaluated in this study. First, the ammonium sulfate precipitation was deployed with supernatant. The saturation of ammonium sulfate, containing 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%, was applied. Subsequently, the ion exchanger column (HiTrap SP HP, 5 mL) was applied to obtain purified pectate lyase using low-pressure liquid chromatography (2001-A-I, Shanghai Jiapeng). Then, liner gradient elution was carried out at 0.8 mL/min, and collection was carried out using a separator. After that, the sample was purified through Sephadex G-75 with pH 7.2 (Tris-HCl, 50 mmol/L) at 0.3 mL/min. Each tube was collected after 10 min.

Protein content was measured by BCA (bovine serum albumin) standard curve. The weight and purity of pectate lyase were measured using SDS-PAGE (X. Zheng et al., 2020).

2.7 Identification of pectate lyase through LC-MS/MS

The signal bond was cut off from the SDS-PAGE gel and subjected to LC-MS/MS (Vogeser and Parhofer, 2007) analysis by Beijing BGI Technology Co., Ltd. The top 23 charge ions from each scan were selected for MS/MS (tandem-mass spectrometry) analysis. Subsequently, MASCOT 2.2 (Matrix Science, London, UK) was applied to search the MS/MS spectrum and identify the protein by searching the Uniport.

2.8 Application of pectate lyase in extraction of apple juice

Two grams of apple pulp were incubated with 5 mL of partially pure pectate lyase for 1 h at 50 °C. Apple juice was acquired by centrifugation at 5,000 rpm for 30 min. After cooling at room temperature, the volume of supernatant and the weight of residue acquired were assessed (Rahman, et al., 2020).

3.1 Screening of strains for pectate lyase

Through the primary and secondary screening, five strains were obtained, including CAS-WZS-08, MEI-2–27, MEI-2–29, CAS-MEI-2–33, and CAS-07, showing in Fig. 1. The CAS-WZS-08 strain had a higher Hc value than others and was selected for further studies.

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Fig. 1

Hc value in pectin isolation medium.

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3.2 Phylogenetic tree and characteristics of the strain

Phylogenetic tree of CAS-WZS-08 was shown in Fig. 2. It had 96% similarity to B. altitudinis 41KF2b T. 23 MN543854.1:1–1423. The sequence of the 16S rDNA was deposited in GenBank, with SUB11024406 CAS-WZS-08 OM462373. The physiological and biochemical characteristics were shown in Table 1. L-arabinose, D-mannose, gelatin liquefaction, nitrate reduction, and gram staining were positive, it also could grow under 7% NaCl. Other tests were negative, including V-P, citrate, propionate, D-xylose, and amylolysis. Antimicrobial susceptibility tests demonstrated that CAS-WZS-08 was susceptible to ampicillin, chloramphenicol, kanamycin, and spectinomycin.

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Fig. 2

The phylogenetic tree of Bacillus altitudinis CAS-WZS-08.

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3.3 Growth characteristic of CAS-WZS-08

The strain grows in a range of pH 5.0 to pH 10.0. The highest biomass was 4.75 ± 0.12 at pH 7.0, while it was markedly inhibited at pH 10.0 (Fig. 3a). The influence of temperature shown that the CAS-WZS-08 grown better at 33 °C (Fig. 3b).

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Fig. 3

The growth of CAS-WZS-08. (a). Growth under different pH conditions; (b). Growth under different temperatures.

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3.4 Optimization of fermentation conditions

The results of fermentation parameter were shown in Fig. 4, including the pectin concentration, nitrogen source, nitrogen concentration, medium pH, inoculum size, and culture temperature. Pectate lyase activity was highest (0.064 ± 0.002 U/mL) at 4 g/L pectin than other (Fig. 4a). Yeast extract, with 0.263 ± 0.013 U/mL enzyme activity, was better than the tested media (Fig. 4b). Organic nitrogen sources were better than inorganic nitrogen in the experiments. The enzyme activity was enhanced 4.1-fold compared to that under a 4 g/L pectin concentration. The influence of yeast extract concentration on pectin lyase activity shown that the enzyme activity increased gradually from 4 g/L to 20 g/L. The highest enzyme activity was 0.54 ± 0.017 U/mL at 20 g/L (Fig. 4c). The effects of medium pH shown the enzyme activity reached 0.69 ± 0.040 U/mL at pH 7.0 (Fig. 4d). Subsequently, the influence of inoculum size on enzyme activity was shown in Fig. 4e, and the highest enzyme activity was obtained with 0.79 ± 0.001 U/mL at 2% (v/v). Finally, the influences of culture temperature from 27 °C to 47 °C were shown in Fig. 4f. The enzyme was no significant difference in enzyme activity between 27 °C and 40 °C, otherwise, it was significantly declined with the temperature exceeded 44 °C.

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Fig. 4

The optimum condition of CAS-WZS-08 for pectate lyase activity. (a). Pectin concentration. (b). Nitrogen source. (c). Yeast extract concentration. (d). Medium pH. (e). Inoculum size. (f). Cultivation temperature. Note: Identical letters indicate a lack of significant difference.

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3.5 Enzyme properties

Enzyme properties were shown in Fig. 5. Under the experiment, the pectate lyase increased gradually with increasing temperature until 60 °C (Fig. 5a). The results of different pH buffer on enzyme activity demonstrated that it was an alkaline pectate lyase, with the highest enzyme activity occurred in pH 10.0 (Fig. 5b). The activity was also affected by the category of the buffer. The Tris-HCl was more suitable for pectate lyase activity than KH₂PO₄-NaOH and Glycine-NaOH. Enzyme activity was activated by Mn²⁺, Cu²⁺, Co²⁺, and Ca²⁺. Especially in the Mn²⁺ condition, the enzyme activity was 2.11-fold that of the control. It was inhibited by Fe³⁺, Ba²⁺, and Mg²⁺, especially Fe³⁺, in which the pectate lyase activity was only 18% of the control. K⁺, Zn²⁺, Ni²⁺, and Fe²⁺ have little effect on enzyme activity (Fig. 5c). The enzyme activity was also affected by buffer concentration, and the highest activity occurred at 50 mmol/L (Fig. 5d). Temperature stability demonstrated that it was stable at 30-50 °C, especially since the enzyme activity was not affected at 30 °C and 35 °C after 180 min (Fig. 5e).

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Fig. 5

Pectate lyase properties of CAS-WZS-08. (a). Optimal temperature. (b). Optimal buffer pH. (c). Metal ions. (d). Buffer concentration. (e). Temperature stability. (f). pH stability.

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Pectate lyase has a wide range of stability from pH 4.0–10.0 at 4 °C, maintaining 100% relative enzyme activity after 24 h incubation (Fig. 5f). The studies on the stability of pectate lyase were summarized in Table 2, including isolation and heterogeneous expression in bacteria.

3.6 Purification and characterization of pectate lyase

To obtain purified pectate lyase, ammonium sulfate fractional precipitation, cation exchange column, and Sephadex G-75 were applied (Fig. 6). The pectate lyase activity was markedly decreased when the ammonium sulfate saturation reached 50% in the supernatant (Fig. 6a). When the saturation reached 70%, the pectate lyase activity diminished to 0 in the supernatant, while the relative activity in the corresponding precipitation reached 93.17% (Fig. 6a). Ultimately, the saturation of ammonium sulfate precipitation was determined to be 50%-70% (Fig. 6a). Subsequently, the crude enzyme was further purified by cation exchange column and Sephadex G-75 (Fig. 6b, 6c). The collection tubes were detected through enzyme activity and SDS-PAGE (Fig. 6d). In the purification process, the enzyme activity and protein content were measured as shown in Table 3. The 1.5 L fermentation liquid was obtained under optimal fermentation conditions, with 927.71 U total enzyme activity and 12936.72 μg protein content. After ammonium sulfate precipitation, 50 mL dialysate was obtained with 221.41 U and 245.30 μg protein. After cation exchange column, the total enzyme activity was 65.09 U with 61.33 μg protein content, and the purification factor was 14.80 with 29.39% yield. After Sephadex G-75, 18.54 U total activity was obtained in 7.51 μg protein with 34.41 purification factor. The purification steps were shown in Fig. 6d with ∼ 37 kDa. LC/MS-MS results showed that the molecular weight of the pectate lyase was 40 kDa with the accession NO. Q56806, which was consistent with the SDS-PAGE results. The matched proteins with a prot-score ≥ 32 were selected, and their detail information were listed in Table S1.

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Fig. 6

Purification of pectate lyase from CAS-WZS-08. (a). Ammonium sulfate fractional precipitation. (b). Cation exchange column. (c). Sephadex G −75. (d). SDS-PAGE. M: Marker, 1: Fermentation liquid. 2. Ammonium sulfate precipitation. 3: Cation exchange column. 4. Sephadex G −75.

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3.7 Application of pectate lyase in extraction of apple juice

Many studies have investigated the effect of pectinase on the yield of fruit juice, including diverse of treatment conditions, the enzyme concentration, and the kinds of enzyme. In this work, the apple juice yield (treated by pectate lyase) increased by 8.29% compared with untreated. The enzyme hydrolysis the pectin of cell wall, releasing the sap inside the cells of pulp, increasing the juice yield (Sharma et al., 2017). The results showed that pulp treated with pectate lyase exhibited better pressing characteristics (Table 4). The weight of residue reduced considerably after enzyme therapy of apple pulp as contrasted with controlling. Rahman et al. (2020) reported that apple juice yield increased by 11.54% through the pectinase, secreting from Bacillus subtilis subsp. Makebe et al., (2020) reported the treatment conditions of soursop pulp using pectinase, including enzyme concentration, temperature, and time, with the 75.20% yield. Mohanty et al., (2018) reported that palm was treated with pectinase and cellulase, and the juice yield reached 87.9 ± 0.66% under optimized conditions. To get better result in the latter studies, the treatment conditions of pectate lyase still need more detailed study.

4.1 The production of pectate lyase in strain

The production capacity of screened strain was an important parameter (Rebello et al., 2017). In this study, the fermentation condition for higher pectate lyase activity was determined, with activities ranging from 0.03 ± 0.01 U/mL to 0.79 ± 0.001 U/mL. Nevertheless, compared with previous studies, the production should be optimized for higher yield. A pectin-rich substrate, such as lemon peel, wheat bran, and orange bagasse, should be utilized instead of pure pectin (Muslim, et al., 2015, Ferreira, et al., 2010). Thite et al., (2020) reported the strain to generate the xylanase and pectinase with four different agro-waste biomasses. The pectinase activity was 220–280 units using the citrus peel. Li et al., (2020) reported that the highest yield of pectinase produced by A. niger NRRL 322 was 9.5 U/mL using soybean hull. Jadhav (2019) screened the B. licheniformis UNP-1 with the 55.2 U/mL pectinase activity. However, the studies on pectate lyase production using agricultural wastes or industrial wastes were limited. To obtain a relatively high fermentation yield, the method of response surface design was used. For example, plackett burman design and central composite design were carried out for optimization of pectate lyase production in B. subtilis PB1 with 19.50 ± 0.28 U/ml activity (Zhou et al., 2017). In the latter study, the higher pectate lyase activity from CAS-WZS-08 might be researched using statistical methods.

4.2 Property of the pectate lyase

Temperature and the pH determine the changes in enzyme activity collectively. The collision between enzyme and substrate was encouraged due to the temperature increased, increasing enzyme activity. However, when a certain limitation was reached, the tertiary structure of the protein was damaged, destroying the enzyme activity (Peterson, et al., 2007). Alkaline pectate lyase is generally applied in textile refining, wastewater treatment, and tea or coffee fermentation (Wu et al., 2020; Zheng et al., 2021). The stability of pectate lyase is extremely important for its application. Zhou et al., (2017) reported the pectate lyase maintained 90% relative activity at 50 °C after 2 h. Prajapati et al., (2021) reported the pectate lyase was stable at pH 4.0–10.0, with 80% relative enzyme activity after 3 h. Zhou et al., (2017) isolated B. subtilis PB1 strain at pH 5 ∼ 11 after 2 h. In this study, the pectate lyase has a wide range of stability from pH 4.0–10.0 at 4 °C. The pH stability not only has a great advantage for storage but also has a wide selection in purification. This result is also a basis for researching the mechanism of pH stability to further enhance the stability of industrial enzymes.

Among the different roles that metal ions can play in the catalytic event, the most common is their ability to orient the substrate correctly for the reaction, exchange electrons in redox reactions, and stabilize negative charges (Prejanò, et al., 2020). Bacterial pectate lyase works predominantly requires Ca²⁺ ions, which could acidify the C5 proton of galacturonic acid binding to the + 1 subsite of pectate lyase (Bekli et al., 2019; Zheng, et al., 2021). Ca²⁺ also was important for pectate lyase producing with CAS-WZS-08.