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Article

Cloning, Expression and Characterization of an Alginate Lyase in Bacillus subtilis WB600

by
Kaixuan Zheng
1,
Yaqing Zhu
1,
Zhiqiang An
2,
Jian Lin
1,
Shoushui Shan
1,* and
Hailing Zhang
1,*
1
Department of Biological Engineering, College of Life Sciences, Yantai University, Yantai 264005, China
2
MabPlex International, Ltd., Yantai 264005, China
*
Authors to whom correspondence should be addressed.
Fermentation 2023, 9(2), 144; https://doi.org/10.3390/fermentation9020144
Submission received: 22 December 2022 / Revised: 28 January 2023 / Accepted: 29 January 2023 / Published: 1 February 2023
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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Abstract

:
The aim of this study was to further broaden the heterologous expression of alginate lyase from Vibrio alginolyticus in a Bacillus subtilis expression vector. A B. subtilis WB600/pP43NMK-alg62 strain was constructed. (NH4)2SO4 precipitation and Ni-affinity chromatography were performed to purify the enzyme. We then characterized the enzyme. Its molecular weight was 57.64 kDa, and it worked optimally at 30 °C with a pH of 8.0. Ca2+ markedly enhanced the enzymatic activity of Alg62 while Cu2+ and Ni2+ inhibited its activity. Alg62 had a wide range of substrate specificity, showing high activity toward sodium alginate and polyG. Following optimization of the fermentation process, the optimal conditions for the recombinant expression of Alg62 were as follows: temperature of 37 °C, pH of 7.0, medium consisting of glycerol 15 g/L, yeast powder 25 g/L and K+ 1.5 mmol/L. At these optimal conditions, enzyme activity reached 318.21 U/mL, which was 1.54 times higher than the initial enzyme activity.

1. Introduction

Alginate is a linear polysaccharide composed of β-D-mannuronic acid (M) and C5 epimer α-L-guluronic acid (G) linked via 1,4-glycosidic bonds. It is arranged as poly β-D-mannuronate (polyM), poly α-L-guluronate (polyG) and the heteropolymer (polyMG) [1]. Alginate predominantly exists in the cell wall and cytoplasm of brown algae, comprising approximately 40% of its dry weight [2]. It is the most abundant marine polysaccharide and is gaining wide attention, owing to its broad application prospects in the fields of nutrition, agronomy and pharmacology. Notably, it can be used as edible films, wound dressings, a vehicle for drug delivery, plant growth stimulants and more [3]. However, its applications are limited because of its high molecular weight, low water solubility and poor bioavailability [4]. Therefore, it is important to degrade high-molecular-weight polysaccharides into low-molecular-weight polysaccharides or oligosaccharides to improve their bioavailability and enhance their efficacy. The degradation products of alginate are known as alginate oligosaccharides (AOS) and consist of alginate oligomers containing 2–25 monomers. AOS are used in a wide range of fields, including food and medicine. They play diverse physiological roles, such as the regulation of immune response [5], exerting anti-oxidation [6,7], anti-bacterial [8] and anti-inflammatory activities [9] and promoting plant growth [10]. AOS can be depolymerized using various methods, including physical, chemical and enzymatic methods [11,12,13,14]. However, the disadvantages of physical and chemical methods include difficulty in controlling reaction conditions, complex product separation, low product stability and environmental pollution. Enzymatic degradation has the advantages of suitability for use under mild conditions, controllable reactions, low energy consumption and environmental protection [15]. It is an effective way to produce AOS on an industrial scale.
Alginate lyase can degrade alginate glycosidic bonds via a β-elimination reaction and produce unsaturated oligosaccharides containing double bonds at the non-reducing end [16]. Currently, alginate lyases have a wide range of sources and have been isolated from a variety of organisms, including marine plants, marine mollusks, marine and terrestrial bacteria and some fungi and viruses [17]. Most alginate lyases are derived from marine bacteria, such as Pseudomonas [18], Vibrio [19], Flavobacterium [20] and Pseudoalteromonas [21]. According to the amino acid sequence alignment, alginate lyases can be divided into seven polysaccharide lyase families, including PL5, PL6, PL7, PL14, PL15, PL17 and PL18, which are carbohydrate-active enzymes [22]. These enzymes play an important role in the preparation of biologically active alginate oligosaccharides [23] for use in biotechnological applications. These include the production of bioethanol and preparation of pharmaceutical intermediates [24,25,26]. However, low yields of alginate lyase with low activity are produced by wild-type microorganisms. Moreover, production is difficult to control, which limits its development and application. With advancements in molecular biology, the use of genetic engineering technology has enabled the efficient heterologous expression of alginate lyase. Some alginate lyases have been successfully expressed in Escherichia coli. Notably, a new endo-type alginate lyase gene, tsaly6A, has successfully been cloned from the marine bacterium Thalassomonas sp. LD5 and expressed in E. coli. It has a specific activity of 15,960 U/μmol [27]. E. coli has the advantages of having a clear genetic background, rapid growth and high gene expression. However, it cannot secrete recombinant proteins into the culture medium, and the recombinant protein is easily degraded and contains endotoxin [28].
Compared with E. coli, B. subtilis is capable of non-specifically secreting recombinant proteins using various signal peptides; they are non-pathogenic and devoid of endotoxins. Therefore, it is an ideal expression host for producing various recombinant enzymes as safe for industrial and pharmaceutical applications [29]. However, there are limited reports on the expression of alginate lyase in B. subtilis.
In this study, we aimed to clone the alginate lyase gene alg62 from the marine bacterium Vibrio alginolyticus ATCC 17749 and expressed it in B. subtilis WB600 using modified shuttle plasmid pP43NMK. An additional aim was to characterize the biochemical properties of the recombinantly expressed Alg62 and optimize the culture conditions for optimal production of Alg62.

2. Materials and Methods

2.1. Strains, Plasmid and Mediums

E. coli JM109, B. subtilis WB600 and plasmid pP43NMK were conserved in our laboratory. The strain of E. coli BL21/pET28a-alg62 was kept in our lab and at the China Center for Type Culture Collection (CCTCC No. M2022537). Sodium alginate was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). PolyM and PolyG (purity > 97%) were purchased from Qingdao BZ Oligo Biotech Co., Ltd. (Qingdao, China). The antibiotic Kanamycin was purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Restriction enzymes XmaI and BstBI were purchased from New England Biolabs (Beijing, China) LTD Co., Ltd. (Beijing, China).
The strains were cultivated in Luria–Bertani (LB) broth containing peptone (10 g/L), yeast extract (5 g/L) and sodium chloride (10 g/L) at 37 °C with shaking at 200 rpm. Seed solution was inoculated into the fermentation medium Terrific Broth (TB), consisting of peptone (12 g/L), yeast extract (24 g/L), glycerol (0.4% v/v), KH2PO4 (0.23 g/L) and K2HPO4 (1.25 g/L) and was used to express alginate lyase. Kanamycin (Kan; 50 µg/mL) was added to the LB and TB media. Alginate lyase was expressed in 100 mL of growth media in 300 mL conical flasks at 37 °C for 24 h.

2.2. Cloning and Sequence Analysis of Alginate Lyase Alg62

Primers were designed as follows: F-Alg43 (base sequence: 5′-CCCTTCGAAATGAAGCATATTTTCTTCAAAAGCTTGTTAGCTT-3′) and R-Alg43 (base sequence: 5′-TCCCCCCGGGCTAGTGGTGATGGTGATGATGATGGCCTTGGTACTTACCAAAAGATTGTTTTA-3′). Following digestion of the recombinant plasmid E. coli BL21/pET28a-alg62 with the restriction enzymes XmaI and BstBI, alg62 was amplified by PCR. The alg62 PCR products were also composed of a 6 × His-tag at the C-terminus. Subsequently, the plasmid pP43NMK was digested with XmaI and BstBI, and the purified alg62 fragment was ligated to the XmaI and BstBI sites of the plasmid. The recombinant plasmid was name pP43NMK-alg62. The online pI/Mw tool (https://web.expasy.org/compute_pi/, accessed on 20 September 2022) was used to determine the theoretical molecular weight (Mw) and the isoelectric point (pI) of the enzyme. The SignalP 4.1 server (http://www.dtu.dk/services/SignalP/, accessed on 20 September 2022) was used to predict signal peptides.

2.3. Expression and Purification of Alg62

The pP43NMK-alg62 was transformed into B. subtilis WB600 cells. The transformed cells were stirred at 37 °C in 50 mL LB medium at 220 rpm for 12–14 h. The overnight cultured cells (2% v/v) were inoculated into 100mL of fermentation medium in a 300 mL Erlenmeyer flask, incubated at 37 °C for 24 h and collected by centrifugation at 10,000 rpm for 20 min at 4 °C. The supernatant containing the crude enzyme was subjected to ammonium sulfate fractionation. A saturated ammonium sulfate solution was briefly added to a final concentration of 80%. After incubation at 4 °C for 12 h, the sample was centrifuged at 10,000 rpm, 4 °C for 20 min. The precipitated protein was redissolved in 50 mmol/L phosphate buffer (PB, pH 8.0). The enzyme solution was concentrated, and residual ammonium sulfate was removed using a 10 kDa Amicon filter (Merck, Munich, Germany). The enzyme solution was then concentrated, the concentrated enzyme solution was loaded onto a lysis-buffer-pre-equilibrated His-Tag Purification Resin column (Beyotime Biotechnology, Shanghai, China). The resin was washed with wash buffer (50 mM NaH2PO4, 300 mM NaCl and 2 mM imidazole; pH 8.0), and the bound protein was eluted from the column using elution buffer (50 mM NaH2PO4, 300 mM NaCl and 50 mM imidazole; pH 8.0). The purified recombinant protein was analyzed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

2.4. Enzymatic Activity Assay

Alginate lyase activity was determined using 3,5-dinitrosalicylic acid (DNS) colorimetry [30]. A total of 0.1 mL of the diluted enzyme solution was briefly mixed with 0.9 mL of 50 mol/L phosphate buffer solution containing 1% sodium alginate (pH 8.0). The mixture was incubated at 30 ºC for 20 min. A total of 1 mL DNS was added, and the reaction was stopped by heating in a boiling water bath for 5 min. The solution was immediately cooled to room temperature, and the volume was brought to a final 10 mL. The concentration of reducing sugar was measured at 520 nm. The blank control group consisted of deionized water instead of the enzyme solution. The amount of reducing sugars produced by the reaction was calculated using glucose as the standard. Enzyme activity was defined as the quantity of enzyme needed to catalyze the substrate to produce 1 μg of reducing sugar per minute.

2.5. Effect of Temperature on Enzyme Activity

To determine the effect of temperature on the activity of the recombinant Alg62, its activity was determined over a temperature range of 25 °C to 60 °C, and the highest enzyme activity value was defined as 100%. The enzyme was briefly incubated at different temperatures between 25 °C and 60 °C for 1 h and then rapidly cooled on ice to determine the residual enzyme activity. The initial enzyme activity value was defined as 100%.

2.6. Effect of pH on Enzyme Activity

To explore the impact of pH on enzyme activity, 50 mM buffers at various pH levels (citrate buffer, pH 4.0–5.0; phosphate buffer, pH 6.0–8.0; glycine-NaOH buffer, pH 9.0–11) were added to the enzyme solution. The mixture was incubated for a 12-h incubation period at 4 °C and its remaining enzyme activity was assessed; 100% was used as the upper limit.

2.7. Effects of Metal Ions on Enzyme Activity

The enzyme solution was placed in 5 mM metal ion solution (Na+, K+, Cu2+, Al3+, Fe2+, Ca2+, Co2+, Ni2+, Mg2+ and Zn2+) and incubated for 2 h at 4 °C. Enzyme activity was then determined. Enzyme activity without metal ions was defined as 100%.

2.8. Substrate Specificity

To investigate the substrate specificity of the recombinant alginate lyase Alg62, 0.5% (w/v) sodium alginate, polymannuronate (polyM) and polyguluronate (polyG) were utilized as substrates to test the enzyme activity. The enzymatic activity of sodium alginate as a substrate was defined as 100%.

2.9. Optimization of the Fermentation Conditions for the Recombinant Expression of Alg62

The effects of the fermentation medium formulation and culture conditions during the recombinant expression of alginate lyase Alg62 in B. subtilis WB600 were investigated.
To optimize the carbon source, sodium alginate, glucose, fructose, maltose, starch and sucrose were selected to replace glycerol in the medium, at an initial concentration of 5 g/L. Similarly, corn steep liquor dry powder, beef extract, yeast extract powder, industrial peptone and soybean cake powder at an initial concentration of 20 g/L were used to replace peptone and yeast powder in the medium. The effect of adding metal ions to the medium on the enzymatic activity during fermentation was studied. The metal ions included were 2 mM K+, Na+, Ca2+, Mg2+, Fe3+, Cu2+ and Zn2+.
Moreover, to determine the optimal culture conditions, a single-factor experiment was used to investigate effects of fermentation parameters on production. Four process parameters, including temperature (25 °C, 30 °C, 33 °C, 37 °C and 40 °C), initial pH (4, 5, 6, 7, 8, 9, 10 and 11), inoculum size (0.1%, 0.5%, 1%, 2%, 4% and 6%) and filling volume (40 mL, 70 mL, 100 mL, 130 mL 150 mL) were assayed in sequence. After the optimal parameters of one factor were obtained, they were used for subsequent fermentation research. All tests were performed in triplicate, and the results are given as average values.

3. Results

3.1. Cloning and Sequence Analysis of Alginate Lyase Alg62

The alg62 gene sequence was cloned from the previously engineered E. coli BL21/pET28a-alg62 in our laboratory (Figure 1). The alg62 gene consists of an open reading frame (ORF) of 1566 bp in length, encoding a protein of 521 amino acid residues. The theoretical molecular weight and pI value of this protein were 57.64 kDa and 4.96, respectively. There were 75 positively charged (Asp + Glu) and 54 negatively charged (Arg + Lys) amino acid residues. According to the signal peptide prediction and sequence analysis by SignalP 4.1, alg62, which includes a signal peptide of 17 residues (Met1 to Ala17), belongs to the polysaccharide lyase family 7 protein.

3.2. Expression and Purification of Alg62

The alg62 gene sequence was successfully inserted into the pP43NMK vector using double digestion with restriction enzymes XmaI and BstBI (Figure 2A) for the recombinant expression of the protein.
B. subtilis WB600/pP43NMK-alg62 was cultured at 37 °C with shaking at 220 rpm for 24 h. Alg62 was recombinantly expressed as an extracellular enzyme, and its activity was 206.48 U/mL. After ammonium sulfate precipitation via NTA-Ni Sepharose affinity chromatography, the specific activity of purified enzyme reached up to 888.4 U/mg; on the basis of crude enzyme (102.22 U/mg), it increased 7.7 times. The specific enzyme activity of purified ALYW201 from Vibrio sp. W2 was 876.4 U/mg [31]; the VsAly7D was expressed in E.coli BL21(DE3), and its specific activity was estimated to be 663.0 U/mg [32]; alginate lyase AlgNJ04 cloned from the marine bacterium Vibrio sp. NJ04 exhibited a specific activity of 2416 U/mg [33]. Analysis by SDS-PAGE (Figure 2B) revealed an obvious band in the purified protein at approximately 57 kDa, which was consistent with the theoretical protein molecular weight. This showed that the recombinant protein was successfully expressed in B. subtilis WB600.

3.3. Effects of Temperature on the Recombinant Alginate Lyase

The optimal temperature for Alg62 was investigated over a temperature range of 20 °C to 65 °C. The relative activity of the enzyme was highest at 30 °C (Figure 3A), which is the same as that of alginate lyases from marine Vibrio sp. OU02 [34], Microbulbifer sp. SH-1 [35] and Vibrio sp. W2 [31]. When the temperature was lower than 30 °C, the relative enzyme activity was maintained at 60%. However, when the temperature was higher than 30 °C, the relative enzyme activity decreased significantly—only 41% at a temperature of 65 °C. The results showed that Alg62 with a lower optimal temperature which can reduce energy consumption and the risk of contamination [27]. After being incubated at 30 °C and 60 °C for 1 h, Alg62 preserved more than 90% and 60% of its activity, respectively; this confirmed that Alg62 was remarkably stable at lower temperatures.

3.4. Effects of pH on Recombinant Alginate Lyase

The optimum pH for Alg62 was studied at various pH levels (4.0–11.0) at 30 °C. The relative enzymatic activity of Alg62 was highest at pH 8.0 (Figure 3B). When the pH reached 11.0, more than 90% of the enzymatic activity was maintained; however, when the pH was less than 8.0, the relative enzymatic activity decreased significantly. These results indicate that the recombinant alginate lyase Alg62 showed good activity in degrading alginate under alkaline conditions. These findings are similar to the properties of some alginate lyases [11,27,36]. The characterization of some alginate lyases are shown in Table 1. Generally, some alginate lyases derived from bacteria prefer to catalyze the hydrolysis reactions in neutral and slightly alkaline pH ranges (Table 1).

3.5. Effects of Metal Ions on the Recombinant Alginate Lyase

The effect of different metal ions on the enzymatic activity of Alg62 is shown in Figure 3C. Na+, K+, Ca2+ and Al3+ promoted enzyme activity. Ca2+ increased enzyme activity the most, whereby the relative enzyme activity reached 117%. This could be attributed to the fact that Ca2+ plays a catalytic role away from the active center and has an important connection with the structural stability of the enzyme [40]. Other ions inhibited enzyme activity, including the heavy metal ion Cu2+, whereby the relative enzyme activity was retained at only 11%, followed by Ni2+ at 27.22%. These results indicate that Alg62 resisted the effects of many metal ions, allowing it to display high activity in an environment containing various ions. Previous investigations have reported that many metal ions can increase enzyme activity (Table 1). For example, the relative enzymatic activities of AlgNJ04 reached 136.2% and 125.32% following activation with 1mM K+ and Ca2+ [33], respectively, with the highest activity observed for AlgL17 in the presence of 0.7 M NaCl, where it reached 170% [41]. However, 1mM Mn2+ and Co2+ can significantly promote the enzymatic activity of Alyw201 [31].

3.6. Substrate Specificity

The substrate specificity of Alg62 was explored using 0.5% (w/v) sodium alginate, polyM and polyG as substrates. Alg62 showed enzymatic activity with all the substrates (Figure 3D). Compared with sodium alginate, the relative enzymatic activity of Alg62 was 98% with polyG, while it was only 40% with polyM, indicating that Alg62 has broad substrate specificity and is a bifunctional lyase. The substrate specificity of PL7 alginate lyase is related to the conserved QIH protein region. AlgNJ-04 and ALW1 alginate lyases show similar activities to sodium alginate and polyG, but lower activity toward polyM [33,37].

3.7. Optimization of Medium Components for Recombinant Alg62 Expression in B. subtilis WB600

3.7.1. Effects of Carbon Sources

The effect of different carbon sources on enzyme production in B. subtilis WB600 was investigated. Enzyme activity was the highest when glycerol was the sole carbon source (Figure 4A), followed by sucrose, glucose, sodium alginate and starch. When glycerol was added at 15 g/L, enzyme activity reached 258 U/mL (Figure 4B); however, enzyme activity gradually decreased as the concentration of glycerol increased. It has been reported that excessive glycerol concentration affects glycerol metabolism and thus affects host growth and enzyme production [42]. Therefore, glycerol, with a mass fraction of 15 g/L, was selected as the carbon source for the fermentation medium.

3.7.2. Effect of Nitrogen Sources

Six different nitrogen sources at 20 g/L were selected to explore their effects on enzyme yield. Enzyme activity and bacterial growth were highest when yeast powder was used as the nitrogen source (Figure 4C). Therefore, it was selected as the nitrogen source for fermentation optimization. Moreover, bacterial growth and enzyme production increased in a dose-dependent manner. Enzyme activity reached a maximum of 279 U/mL when the concentration of yeast powder was 25 g/L, (Figure 4D). Therefore, yeast powder at a concentration of 25 g/L was selected as the nitrogen source for fermentation.

3.7.3. Effect of Adding Metal Ions

Metal ions play an indispensable role in bacterial growth and enzyme production; they can act as cofactors to promote enzyme activity [43]. Different types and concentrations of metal ions were investigated on their effect on Alg62 yield. Bacterial growth was highest when Mg2+ was added with an OD600 reaching 9.5. However, enzyme activity was highest when K+ was added (Figure 4E). The main purpose of this study was to improve enzyme activity; therefore, K+ was selected as the additional metal ion in the fermentation medium. We then optimized the concentration of K+ and found that enzyme activity (302.46 U/mL) was highest at a concentration of 1.5 mM (Figure 4F). Therefore, this concentration of K+ was selected as the metal ion in the fermentation medium.

3.8. Optimization of Fermentation Conditions for Recombinant Alg62 Expression in B. subtilis WB600

3.8.1. Effect of Fermentation Temperature

Temperature is an important factor affecting bacterial growth and metabolism. Additionally, enzymes are sensitive to temperature. The effects of temperature between 25 °C and 40 °C on the recombinant expression of Alg62 was investigated. It was observed that with an increase in temperature, the activity and cell growth of Alg62 gradually increased (Figure 5A), and both reached an optimum at 37 °C, with an enzyme activity of 308.57 U/mL. However, when the temperature exceeded 37 °C, both enzyme activity and cell growth declined. Therefore, the optimum fermentation temperature was 37 °C.

3.8.2. Effect of Initial pH

The initial pH of the medium affects the permeability and stability of the cell membrane, thereby affecting the absorption of nutrients by the cells and consequently enzyme production and activity. Therefore, the effect of different pH values (4.0–11.0) on the activity of Alg62 was investigated (Figure 5B). When the pH was between 7.0 and 8.0, bacterial growth was good; however, it was best at pH 8.0. At pH 7.0, the activity of Alg62 was highest, reaching 312.26 U/mL. At other pH values investigated, both bacterial growth and enzymatic activity are inhibited, possibly because of altered enzyme structures [44]. Therefore, the initial pH of fermentation was selected as 7.0 for further experiments.

3.8.3. Effect of the Inoculum Size

The amount of inoculum had a significant influence on the duration of the fermentation process and bacterial growth. An optimal amount of inoculum can shorten the growth cycle and improve enzyme production efficiency. The inoculum amount was investigated within a range of 0.1–6% (v/v) (Figure 5C). When the inoculation was between 0.1–4%, cell growth and enzyme production increased gradually; however, they were highest at an inoculation of 4%, where the enzyme activity was 314.42 U/mL. When the inoculation amount was 6%, both enzyme activity and cell growth decreased slightly. The optimal inoculation was found to be 4%.

3.8.4. The Effect of Filling Volume

The filling volume has a direct impact on the amount of dissolved oxygen in the fermentation process and thus impacts bacterial growth and enzyme production. If the oxygen content is too high, oxygen-containing free radicals can easily arise that destroy cell components, thus seriously affecting the growth and metabolism of bacteria [45]. The optimization results for the filling volume are presented in Figure 5D. When the filling volume was 130 mL/300 mL, cell growth was optimal, and the enzyme activity was 318.21 U/mL. With an increase in the filling volume, both cell growth and enzyme activity started to decrease, indicating that the amount of dissolved oxygen started to decrease.

4. Conclusions

In this study, a cold-adapted alginate lyase of Alg62 was successfully recombinantly expressed in B. subtilis WB600. Recombinant Alg62 showed the highest activity at 30 °C, pH 8.0 and 50 mmol/L Ca2+. Most of the reported alginate lyases from the PL7 family share characteristics similar to those of Alg62, such as AlyA, AlyB, AlyD and AlyE, with optimal activity in the pH 7.5 to 8.5 and 20 °C to 25 °C ranges [46]; VsAly7D showed highest activity under alkaline conditions (pH 8.0) at 35 °C [32]. Meanwhile, substrate specificity analysis revealed Alg62 activities toward sodium alginate, polyG and polyM, indicating that the enzymes were a bifunctional alginate lyase. Because Alg62 is cold-adapted, is pH-stable and has various metal ion-resistance properties, it can be efficiently applied to the industrial production of oligosaccharides.
Simultaneously, an engineered strain, B. subtilis WB600/pP43NMK-alg62, was constructed without the need for induced expression and cell disruption. The fermentation process was optimized by single-factor experiments, and optimal fermentation conditions were determined as follows: glycerol 15 g/L, yeast powder 25 g /L, K+ 1.5 mM, 37 °C, pH 7.0, 4% inoculum volume and 130 mL/300 mL liquid volume; after 24 h of shake-flask fermentation, the enzymatic activity of algin lyase increased from 206.48 U/mL to 318.21 U/mL. This lays the foundation for the efficient expression of alginate lyase in Bacillus subtilis and has broad implications for the preparation and application of alginate oligosaccharides [38].

Author Contributions

Conceptualization, K.Z. and H.Z.; methodology, K.Z. and Y.Z.; software, K.Z.; validation, S.S. and J.L.; formal analysis, Z.A.; investigation, K.Z. and S.S.; resources, J.L.; data curation, J.L. and S.S.; writing—original draft preparation, K.Z. and H.Z.; writing—review and editing, H.Z. and S.S.; visualization, Z.A.; supervision, H.Z.; project administration, J.L and S.S.; funding acquisition, H.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (No. 32001836), the Natural Science Foundation of Shandong Province (ZR201911180224) and the Yantai Science and Technology Development Plan (SK21H266).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Fermentation 09 00144 g001 550
Figure 1. PCR product of alg62. M: 2000bp DNA marker; lane 1: amplification product of alginate lyase.
Figure 1. PCR product of alg62. M: 2000bp DNA marker; lane 1: amplification product of alginate lyase.
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Figure 2. (A) Analysis of the double digestion of the recombinant plasmid. M: 10,000 bp DNA marker; lane 1: double digestion with empty plasmid pP43NMK; lane 2: double digestion with recombinant plasmid pP43NMK-alg62. (B) SDS-PAGE of the recombinant Alg62. M: protein marker; lane 1: fermentation supernatant; lane 2: (NH4)2SO4 precipitate; lane 3: purified Alg62.
Figure 2. (A) Analysis of the double digestion of the recombinant plasmid. M: 10,000 bp DNA marker; lane 1: double digestion with empty plasmid pP43NMK; lane 2: double digestion with recombinant plasmid pP43NMK-alg62. (B) SDS-PAGE of the recombinant Alg62. M: protein marker; lane 1: fermentation supernatant; lane 2: (NH4)2SO4 precipitate; lane 3: purified Alg62.
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Figure 3. Biochemical characterization of Alg62. (A) Optimum temperature and thermostability of Alg62. The squares represent the optimum temperature, and the triangles represent the thermostability. (B) Optimum pH and pH stability of Alg62. The squares represent the optimum pH, and the triangles represent the pH stability. (C) Effects of metal ions on Alg62. (D) Substrate specificity of Alg62. The data are presented as the mean ± standard deviation of three assays.
Figure 3. Biochemical characterization of Alg62. (A) Optimum temperature and thermostability of Alg62. The squares represent the optimum temperature, and the triangles represent the thermostability. (B) Optimum pH and pH stability of Alg62. The squares represent the optimum pH, and the triangles represent the pH stability. (C) Effects of metal ions on Alg62. (D) Substrate specificity of Alg62. The data are presented as the mean ± standard deviation of three assays.
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Figure 4. Optimization of medium components for recombinant expression of Alg62 in B. subtilis WB600. The histogram represents the enzyme activity of Alg62 and the line graph represents bacterial growth. (A) Effect of carbon sources on enzyme activity and cell growth. (B) Effect of glycerol concentration on enzyme activity and cell growth. (C) Effect of nitrogen sources on enzyme activity and cell growth: 1, peptone; 2, yeast powder; 3, corn steep liquor dry powder; 4, beef extract; 5, yeast extract powder; 6, soybean cake powder; 7, industrial peptone. (D) Effect of concentration of yeast powder on enzyme activity and cell growth. (E) Effect of metal ions on enzyme activity and cell growth. (F) Effect of K+ concentration on enzyme activity and cell growth. The data are presented as mean ± standard deviation of three assays.
Figure 4. Optimization of medium components for recombinant expression of Alg62 in B. subtilis WB600. The histogram represents the enzyme activity of Alg62 and the line graph represents bacterial growth. (A) Effect of carbon sources on enzyme activity and cell growth. (B) Effect of glycerol concentration on enzyme activity and cell growth. (C) Effect of nitrogen sources on enzyme activity and cell growth: 1, peptone; 2, yeast powder; 3, corn steep liquor dry powder; 4, beef extract; 5, yeast extract powder; 6, soybean cake powder; 7, industrial peptone. (D) Effect of concentration of yeast powder on enzyme activity and cell growth. (E) Effect of metal ions on enzyme activity and cell growth. (F) Effect of K+ concentration on enzyme activity and cell growth. The data are presented as mean ± standard deviation of three assays.
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Figure 5. Optimization of fermentation conditions for recombinant production of Alg62 by B. subtilis WB600. The histogram represents the activity of Alg62 and the line graph represents cell growth. (A) Effect of temperature on enzyme activity and cell growth. (B) Effect of initial pH on enzyme activity and cell growth. (C) Effect of inoculum size on enzyme activity and cell growth. (D) Effect of filling volume on enzyme activity and cell growth. The data are presented as mean ± standard deviation of three determinations.
Figure 5. Optimization of fermentation conditions for recombinant production of Alg62 by B. subtilis WB600. The histogram represents the activity of Alg62 and the line graph represents cell growth. (A) Effect of temperature on enzyme activity and cell growth. (B) Effect of initial pH on enzyme activity and cell growth. (C) Effect of inoculum size on enzyme activity and cell growth. (D) Effect of filling volume on enzyme activity and cell growth. The data are presented as mean ± standard deviation of three determinations.
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Table
Table 1. Characterization of some reported alginate lyases.
Table 1. Characterization of some reported alginate lyases.
EnzymeOriginMolecular Weight (kDa)Optimum pH/Temperature (°C)Substrate
Specificity		Activating CationReference
AlyA-OU02Vibrio sp. OU02658.0/30polyMFe2+, Mg2+[34]
TsAly6AThalassomonas sp. LD5848.0/35Alginate, polyGCa2+, Mg2+[27]
AlgBVibrio sp. Ni1678.0/35polyM, AlginateK+[36]
AlgNJ04Vibrio sp. NJ-04507.0/40polyGCa2+, K+, Na+[33]
ALW1Microbulbifer sp. ALW1267.0/45Alginate, polyGNa+[37]
AlgSH7Microbulbifer sp. SH-166.49.0/40polyMNa+, K+, Al3+, Fe3+[38]
AlgSH17Microbulbifer sp. SH-182.547.0/30polyMMg2+, Fe2+, Mn2+[35]
Alyw201Vibrio sp. W2388.0/30/Mn2+, Co2+[31]
AlgM4Vibrio sp. M0101558.5/30Alginate, polyG, polyMMg2+, Na+[39]
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Zheng, K.; Zhu, Y.; An, Z.; Lin, J.; Shan, S.; Zhang, H. Cloning, Expression and Characterization of an Alginate Lyase in Bacillus subtilis WB600. Fermentation 2023, 9, 144. https://doi.org/10.3390/fermentation9020144

AMA Style

Zheng K, Zhu Y, An Z, Lin J, Shan S, Zhang H. Cloning, Expression and Characterization of an Alginate Lyase in Bacillus subtilis WB600. Fermentation. 2023; 9(2):144. https://doi.org/10.3390/fermentation9020144

Chicago/Turabian Style

Zheng, Kaixuan, Yaqing Zhu, Zhiqiang An, Jian Lin, Shoushui Shan, and Hailing Zhang. 2023. "Cloning, Expression and Characterization of an Alginate Lyase in Bacillus subtilis WB600" Fermentation 9, no. 2: 144. https://doi.org/10.3390/fermentation9020144

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