A cold-adapted and detergent-stable α-amylase from marine bacterium Photobacterium gaetbulicola Gung47^T

Abstract

Abstract

A novel cold-adapted and halotolerant α-amylase gene (AmyPG2) was cloned and expressed from the marine bacterium Photobacterium gaetbulicola Gung47^T. The highest activity of AmyPG2 was displayed at 25 °C and pH 8.0. In addition, the residual activity of AmyPG2 remained at approximately 12–30% at low temperatures (0–5 °C). It can remain 50% activity after 1.6 and 1.0 h at 35 and 40 °C, respectively, demonstrating remarkable thermal stability among cold-adapted enzymes. AmyPG2 was strongly stimulated by NaCl, with its specific activity towards various substrates increasing more than 100-fold. In particular, the specific activity towards mung starch reached 1352.2 ± 45.7 U/mg. The catalytic efficiency was further improved approximately 2.5-fold by site-directed mutagenesis near the putative binding sites. AmyPG2 and its mutant I236V were able to efficiently saccharify starch at low temperature (25 °C), achieving the final hydrolysis rates of 51.2 ± 1.8 and 62.5 ± 2.4% for 8% mung starch, respectively. In addition, AmyPG2 and I236V showed good tolerance to all commercial detergents tested, significantly improving the detergent removal efficiency. This study demonstrated the potential of the cold-adapted α-amylase AmyPG2 and its mutant for industrial applications, particularly in food processing and detergent formulation.

Key points

• A novel α-amylase (AmyPG2) from a marine bacterium was cold-adapted and halotolerant.

• The I236V mutant (2.5-fold higher activity) was obtained by site-directed mutagenesis.

• The enzyme has potential applications in starch saccharification and detergent formulation.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00253-026-13811-3.

Introduction

Marine microorganisms growing in harsh environmental conditions represent a plentiful source for exploring novel biocatalysts with distinctive properties, including cold adaptivity, high salt tolerance, and barophilicity. Cold-adapted enzymes are able to function effectively under low-temperature conditions; therefore, they are suitable for industrial processes conducted at room temperature or in refrigerated environments. For example, the β-galactosidase from Pseudoalteromonas haloplanktis can efficiently hydrolyze lactose under refrigerated conditions, simplifying the industrial production of lactose-free products (Van De Voorde et al. 2014). The protease from Pseudoalteromonas sp. SM9913 improved meat quality by tenderizing at cold storage conditions (4 °C) while effectively preserving moisture and maintaining bright colors (Zhao et al. 2012). Cold-adapted enzymes are defined by an optimal temperature of activity that is typically between 0 and 30 °C, along with high thermal instability (Gerday et al. 1997). However, cold-adapted enzymes meeting this definition are rare. Many enzymes described as cold-active actually have optimum temperatures at 30–45 °C, exhibiting low activity at 5–20 °C (Bendtsen et al. 2025).

α-Amylases (EC 3.2.1.1) are versatile hydrolases, accounting for approximately 30% of the global share in the enzyme industry (Wang et al. 2018; Paul et al. 2021). They randomly cleave α−1,4-glycosidic bonds inside starch and are broadly employed in various manufacturing fields such as food, starch syrups production, detergents, and desizing industries (Far et al. 2020). Cold-adapted α-amylase supports low-temperature processing, thereby preserving heat-sensitive components and ensuring product quality. Thus, the enzyme has been employed in a range of novel applications. For example, in the fruit juice processing industry, to prevent juice spoilage and nutrient loss, fruit juice is treated at low temperature. A cold-adapted α-amylase from Cladophora hutchinsiae was used to reduce the viscosity and turbidity in chilled juice (Cakmak et al. 2022). The laundering of textiles containing wool and silk requires temperatures below 30 °C to avoid damage to the protein fibers. Cold-adapted α-amylases from Bacillus cereus GA6 and Pseudoalteromonas sp. M175 were added to detergents for laundering at room temperature (22–25 °C) (Kuddus 2013; Wang et al. 2018). However, a significant number of α-amylases discovered are thermo- or mesophilic. Just a few exhibit optimal activity at or below 25 °C (Feller et al. 1992; Wang et al. 2018; Sanchez et al. 2019; Kizhakedathil 2021; Ariaeenejad et al. 2021; Cakmak et al. 2022). Additionally, some salt-tolerant α-amylases have been reported to have good compatibility with detergents and have great potential for use in detergents (Kanthi Kiran and Chandra 2008; Chakraborty et al. 2011; Moshfegh et al. 2013; Kalpana and Pandian 2014). Textiles in special industries contain high salt concentrations, which require salt-tolerant enzymes to completely remove stains (Wang et al. 2018). However, there is very limited research on α-amylases that possess both cold-adapted (25 °C or lower) and salt-tolerant properties (Ariaeenejad et al. 2021; Wang et al. 2018; Dou et al. 2018).

Photobacterium gaetbulicola Gung47^T (=KCTC 22804^T =CCUG 58399^T) is a marine bacterium known to hydrolyze starch, yet it remains seldom explored (Kim et al. 2010). Whole genome sequencing reveals that the strain possesses at least three putative amylase genes. Previously, we cloned and characterized an α-amylase (AmyPG) from this strain, which belongs to GH13_37 (Fu et al. 2024). The other two, AmyPG2 and AmyPG3 (GenBank ID AJR06082 and AJR06406), which belong to the GH13_27 subfamily, were obtained as recombinant proteins in E. coli. AmyPG3 was identified as existing in the form of inclusion bodies. Various strategies, including expression optimization and in vitro renaturation, were unsuccessful. Therefore, this study investigates the biochemical properties of AmyPG2. Its catalytic efficiency was further improved by site-directed mutagenesis. Furthermore, the essential applications of AmyPG2 and its mutant were additionally explored as starch-saccharifying enzymes and laundry detergent additives.

Materials and methods

Cloning, expression, and purification of recombinant AmyPG2

The AmyPG2 gene (Sequence S1) obtained from the full genomic sequence of the marine bacterium P. gaetbulicola Gung47 was designed with E. coli codon optimization and constructed by Shanghai Sangon Biotech Co., Ltd. Through standard protocols, the synthetic gene was inserted between the BamHI and XhoI sites of pET28a to obtain pET28a-AmyPG2. Subsequently, heterologous expression was achieved by transforming it into E. coli BL21 (DE3) (Novagen). In Luria-Bertani (LB) medium containing 50 µg/mL kanamycin, the transformant was cultured at 37 °C to an OD₆₀₀ of 0.7. Following induction with 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG), the culture was cultured for another 8 h at 16 °C. Purification of polyhistidine-tagged recombinant AmyPG2 was performed using a HisTrap™ HP column (GE Healthcare) via affinity chromatography following the supplier’s instructions. AmyPG2 was subjected to SDS-PAGE for molecular mass analysis and to the Bradford method for concentration determination.

Amylase assay

α-Amylase activity was quantified by measuring the content of reducing sugars, using the 3,5-dinitrosalicylic acid (DNS) method (Miller 1959). The reaction mixture, consisting of 1 mL of 2% soluble starch (or other substrates) and 200 µL of suitably diluted enzyme in 50 mM phosphate-citric acid buffer (pH 8.0), was incubated at 25 °C for 10 min. The reaction mixture with an equal volume of buffer instead of the enzyme solution served as a blank control. Gelatinized starch solution was prepared from raw starch according to Baks et al. (2008) by heating at 90 °C for 1 h. One unit of activity (U) was defined as the amount of enzyme needed to release 1 µmol of reducing sugar (expressed as glucose) per minute.

Biochemical characterization of AmyPG2

The optimal temperature for the hydrolytic activity of purified AmyPG2 was identified over the range 0–45 °C in 50 mM phosphate-citric acid buffer (pH 8.0). The enzyme activity at 25 °C was set to 100%. Thermostability was measured by pre-incubating the enzyme at 20, 25, 30, 35, and 40 °C without substrates. The activity was designated as 100% before incubation. Aliquots were removed at predetermined time points and assayed at 25 °C following the aforementioned method. To identify the optimal pH, activities were quantified using 50 mM phosphate-citrate (pH 4.0–8.0) and glycine-NaOH (pH 8.0–10.5) buffers. The maximum activity observed at pH 8.0 was set as 100%. The pH stability was assessed through subjecting AmyPG2 to varying pH conditions for 1 h at 4 °C. The activity was designated as 100% before incubation. Under standard conditions (25 °C, pH 8.0), the retained activity was quantified.

The influence of varying metal ions on AmyPG2 activity was evaluated by testing it with 5 mM MgCl₂, MnCl₂, KCl, ZnSO₄, BaCl₂, CuSO₄, FeCl₃, and CaCl₂ under standard conditions. The enzymatic activity without addition was designated as 100%. To study the influence of NaCl on AmyPG2, activities were assayed in the 0–4 M NaCl range. The activity without additives was taken as 100%.

AmyPG2 substrate specificity was evaluated in the presence and absence of 2.0 M NaCl. The corresponding enzyme-free reaction systems were used as blank controls. The substrates measured included pullulan, soluble starch, β-cyclodextrin, α-cyclodextrin, amylose, γ-cyclodextrin, amylopectin, and five gelatinized starches (mung, wheat, corn, potato, and rice). The substrate concentrations were 2% and 5% for various polysaccharides and the gelatinized starches, respectively. The reaction system without the enzyme was used as a blank control.

For kinetic analysis, enzyme activities were also analyzed with and without 2.0 M sodium chloride, with the corresponding enzyme-free reaction systems used as blank controls. The concentration range of soluble starch was 20–300 mg/mL under salt-free conditions, while the range was reduced to 5–80 mg/mL in salt-supplemented reactions. The parameter data were derived from the Lineweaver-Burk plot.

Analysis of hydrolysis products

Thin-layer chromatography (TLC) was used to identify the soluble starch hydrolysates. In a final volume of 10 mL, 5 units of AmyPG2 were reacted with 2% soluble starch at 25 °C. Samples were removed after 0.5, 1, 3, and 8 h of reaction and then boiled in water for 10 min. Products of hydrolysis were resolved using a silica gel G-60 plate. Following development of the hydrolysate with isopropanol-ethyl acetate-water (3:1:1, v/v/v), the plate was sprayed with a methanol solution including N-(1-naphthyl)-ethylenediamine (0.3%) and H₂SO₄ (5%, v/v) and then heated for 10 min at 110 °C to render the spots visible (Liu et al. 2012).

Site-directed mutagenesis of AmyPG2

Using the AlphaFold system to forecast a highly credible structural model of AmyPG2 (Jumper et al. 2021), PROCHECK was used to evaluate the stereochemical quality of the 3D structure of AmyPG2 via the SAVESv6.0 toolkit (https://saves.mbi.ucla.edu/). Acarbose was selected as the ligand molecule, and its 3D structure was generated using the website of PubChem (https://pubchem.ncbi.nlm.nih.gov). The semi-flexible docking of AmyPG2 and acarbose was achieved using the AutoDock tool v1.5.7, and the conformations were visually analyzed with PyMOL software. Based on the results of docking analysis, possible binding sites were selected for site-directed mutation.

Mutagenesis of the pET28a-AmyPG2 plasmid was performed employing the TaKaRa MutanBEST Kit (TaKaRa, China) with the aim of obtaining potential mutants. All used primers are provided in Table S1. After confirmation by sequencing (Tongyong, China), the successfully sequenced mutants were transferred to E. coli BL21, and subsequent operations were carried out.

Starch degradation

Starch slurry (8%, mung and wheat) was heat-treated to gelatinize. 2 M NaCl to this reaction was added. The well-established commercial enzyme, porcine pancreatic α-amylase (PPA, Sigma Chemical Co., China), was used as the control. The dosage of AmyPG2, I236V, and PPA was 1.0 U/mg dry starch. A total reaction mixture was brought up to 8 mL with buffer, with the addition of 0.2% (v/v) toluene to prevent microbial growth (Hamilton et al. 1998). PPA was used at pH 7.0 in a 50 mM phosphate buffer with 5 mM CaCl₂. Then, the reaction solutions of these three enzymes were all reacted for 16 h at 25 °C. Aliquots were taken at appropriate time points, after which the reaction was terminated by mixing with 0.3 M Na₂CO₃ stop solution. The content of reducing sugars released during the reaction was assayed using the DNS method.

Detergent compatibility with various commercial laundry detergents

The stabilities of the wild-type AmyPG2 and I236V mutant were evaluated with various Chinese commercial liquid detergents. The detergents tested were Baimao^®, Blue Moon^®, Chaoneng^®, Diaopai^®, Liby^®, OMO^®, and Walch^®. To denature any endogenous enzymes, all commercial detergents (1%, v/v) were subjected to heat inactivation at 100 °C for 1 h to eliminate their activity. The purified enzyme was then mixed with each detergent solution and incubated at 25 °C for 1 h. Remaining activities were quantified under standard assay conditions. A detergent-free control sample incubated in identical conditions was defined as 100%.

Wash performance analysis

Stain removal analysis was carried out and modified according to the protocol of Roy et al. (2012). Liquefaction of chocolate was achieved by heating at 70 °C. Cotton fabrics were stained with 100 µL of the chocolate or tomato sauce (starch-rich), respectively, and then dried overnight in an oven. To evaluate washing performance, each stained fabric sample was incubated separately at 25 °C for 1 h in 25 mL of one of three solutions: (a) distilled water containing 2 M NaCl, (b) a mixture of 20 mL distilled water containing 2 M NaCl and 5 mL of detergent (1%, v/v), or (c) the same detergent mixture supplemented with the enzyme at 0.4 U/mL. After incubation, all fabrics were rinsed with water and air-dried prior to analysis.

Statistical analysis

All experiments were performed in triplicate, and the results were expressed as mean ± standard deviation (SD). Statistical analysis was performed using Student’s t-test. A p value less than 0.05 was considered statistically significant.

Results

Expression and purification of the recombinant AmyPG2

From the whole genome of P. gaetbulicola Gung47, we identified an α-amylase, named AmyPG2. The enzyme consists of 462 amino acids, consistent with a predicted molecular weight of 50.8 kDa. Within the CAZy database (http://www.cazy.org/), AmyPG2 is assigned to the subfamily GH13_27, which consists of only bacterial α-amylases. The AmyPG2 sequence contains all five conserved sequence regions (CSRs) of GH13_27 (Fig. S1). Up to now, seven characterized α-amylases of GH13_27 have been reported (Kang et al. 2001; da Silva et al. 2002; Allala et al. 2019; Hu et al. 1992; Han et al. 2014; Wu et al. 2017; Gobius and Pemberton 1988). AmyPG2 showed the highest sequence identity (54.1%) to an α-amylase Amy3 from Pseudomonas sp. KFCC10818 (Kang et al. 2001).

The recombinant AmyPG2 was solubly expressed in E. coli BL21 (DE3). Additionally, it was purified using a Ni²⁺-chelating column. The molecular mass estimated from SDS-PAGE was consistent with the theoretical value calculated from the protein sequence (Fig. S2a).

Biochemical characterization of AmyPG2

The purified AmyPG2 displayed the optimal activity at 25 °C (Fig. 1a), with over 90% of the highest activity retained within the 20–30 °C range. AmyPG2 was also highly active at lower temperatures, with approximately 12 and 30% of its highest activity maintained at 0 and 5 °C, respectively. The data suggested that AmyPG2 was a cold-adapted enzyme with strong adaptation to cold temperatures. The half-lives of AmyPG2 were determined to be approximately 10, 6.6, 4, 1.6, and 1.0 h at 20, 25, 30, 35, and 40 °C (Fig. 1b). The optimal pH for AmyPG2 activity was 8.0 (Fig. 1c), and the enzyme showed relatively high stability across the 6.0–10.0 range. Remarkably, it could still keep approximately 74, 57, and 49% of the highest activity at pH 9, 10, and 10.5. AmyPG2 showed high stability over pH 7.0–11.0, maintaining over 89% of its original activity after 1 h at 4 °C (Fig. 1c). Almost no activity loss was observed at pH 8.0. Therefore, AmyPG2 was identified as an alkaline amylase.

Fig. 1.

Effects of temperature and pH on the activity and stability of AmyPG2. a Effect of temperature on enzyme activity. b Effect of temperature on the enzyme stability: (square) 20 °C, (circle) 25 °C, (up-pointing triangle) 30 °C, (diamond) 35 °C, and (down-pointing triangle) 40 °C. c Effect of pH (black) and stability (blue) on enzyme activity: (white square) 50 mM phosphate-citric acid buffer pH 4.0–8.0 and (black square) 50 mM glycine-NaOH buffer pH 8.0–11.0. The results were expressed as mean ± standard deviation (SD)

Effects of NaCl and metal ions on enzyme activity

Analysis of the effect of NaCl (0–4.0 M) on the activity of AmyPG2 is presented in Fig. 2a. AmyPG2 had only the specific activity of 4.2 ± 0.08 U/mg in 50 mM glycine-NaOH buffer without the addition of NaCl, but the activity increased dramatically with NaCl. Indeed, the highest activity was observed at 2.0 M NaCl, with about 115-fold improvement, suggesting that AmyPG2 was a halotolerant enzyme. Moreover, it maintained above 70% of its peak activity with 4.0 M NaCl, demonstrating its high salt tolerance.

Fig. 2.

Effects of 0–4 M NaCl (a) and 5 mM metal ions (b) on the activity of AmyPG2. The results were expressed as mean ± standard deviation (SD)

The effect of different 5 mM metal ions on the enzymatic activity was also determined (Fig. 2b). Ca²⁺, K⁺, and Mg²⁺ all enhanced the activity of AmyPG2. Among them, Mg²⁺ was the most effective, yielding a 1.7-fold increase in activity. Other tested metal ions, such as Ba²⁺, Cu²⁺, Fe³⁺, Mn²⁺, and Zn²⁺, exhibited inhibitory effects on the activity.

Substrate specificity and hydrolysis products

The substrate specificity of AmyPG2 was assessed in the presence and absence of 2.0 M NaCl (Table 1). AmyPG2 was hydrolytically active on soluble starch, amylose, amylopectin, gelatinized mung starch, gelatinized rice starch, gelatinized potato starch, gelatinized corn starch, and gelatinized wheat starch. It showed only weak activity towards these substrates in the absence of NaCl. However, after adding salt to the reaction mixture, the activity improved by approximately 106- to 119-fold. No activity towards pullulan was detected in the absence of NaCl, whereas slight activity (12.9 ± 0.44 U/mg) was observed with NaCl. Additionally, AmyPG2 displayed no activity towards α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, whether NaCl was added or not.

Table 1.

Hydrolysis activities of AmyPG2 on various substrates

Substrates	Specific activity without NaCl (U/mg)	Relative activity without NaCl (%)	Specific activity added 2.0 M NaCl (U/mg)	Relative activity added 2.0 M NaCl (%)
Soluble starch	4.2 ± 0.08	100	484.3 ± 12.3	100
Amylose	3.9 ± 0.17	92.8	425.5 ± 18.7	87.8
Amylopectin	3.1 ± 0.14	73.8	371.3 ± 11.8	69.8
Pullulan	0	/	12.9 ± 0.44	2.7
α-Cyclodextrin	0	/	0	/
β-Cyclodextrin	0	/	0	/
γ-Cyclodextrin	0	/	0	/
Gelatinized starch
Mung	12.7 ± 0.52	302	1352.2 ± 45.7	279
Wheat	9.2 ± 0.31	219	1081.5 ± 24.1	223
Corn	8.5 ± 0.37	202	1011.5 ± 19.8	209
Potato	7.3 ± 0.26	174	824.4 ± 23.0	170
Rice	6.8 ± 0.24	162	743.9 ± 11.3	154

TLC was performed to analyze the hydrolytic soluble starch products (Fig. 3). During the initial phase of hydrolysis, maltose (G2), maltotriose (G3), and maltotetraose (G4) accumulated as the primary products. With extended incubation, maltose and maltotriose increased, whereas maltotetraose was no longer detectable and glucose was not observed at any stage. Therefore, AmyPG2 can be classified as a saccharifying-type α-amylase that generates predominantly low-molecular-weight oligosaccharides, such as disaccharides and trisaccharides, during hydrolysis (Liu et al. 2012; Kanpiengjai et al. 2017).

Fig. 3.

TLC analysis of products resulting from soluble starch hydrolysis. Maltooligosaccharide standards (G1-G4) were marked in lane Std

Preparation of site-directed mutagenesis near the putative binding sites

Since an experimental 3D structure was not found in PDB for any GH13_27 α-amylase, the AlphaFold-predicted structure of AmyPG2 was used to assess the stereochemical quality of the predicted structures. The Ramachandran plot showed that 92.0% of the residues were located in the most favored region, 7.8% in the additionally allowed region, 0.2% in the generously allowed region, and no residues were in the disallowed region (Fig. S3). The overall G factor is −0.06, which falls within the acceptable range (> −0.5). This result confirms the reliability of the structure (Shoaib et al. 2026; Kumar et al. 2023). By docking acarbose with AmyPG2, the binding energy was −8.26 kcal/mol (Table S2), and five amino acid residues (S231, I236, T262, S263, and R302) were selected as mutation sites (Fig. 4a). The corresponding mutants S231A, I236A, T262A, S263A, and R302A were individually created using a one-step PCR mutagenesis. Among these five mutants, I236A showed the highest increase in activity. We also noticed that position 236 in the subfamily GH13_27 is mostly occupied by Val (V) (Fig. 4b). Therefore, the mutant I236V was further constructed.

Fig. 4.

a Substrate-binding area of AmyP2. Bound acarbose is depicted as sticks with gray indicating carbon atoms; the five residues hydrogen-bonded to acarbose are presented in a stick model and marked in blue. The hydrogen bonds are marked with yellow dashed lines. b Sequence logos of α-amylases from GH13_27 subfamily. The logo is based on identifying conserved sequence regions III of GH13 family

The sequences of all mutants were confirmed prior to their expression in E. coli BL21 (DE3). Enzymes were then purified to homogeneity by nickel-affinity chromatography (Fig. S2b).

Characterization of mutants

Compared to the wild-type AmyPG2, the optimal temperature was increased by 5 °C for mutant T262A, while the optimal pH was decreased by 0.5 for mutants S263A and R302A. Other mutants retained the same optimal temperature and optimal pH as AmyPG2 (Table 2). The optimal NaCl concentration remained unchanged at 2 M for all mutants. Meanwhile, NaCl did not affect the relative specific activities of various mutants to soluble starch. Both in the presence and absence of NaCl, the mutants I236A, I236V, and S263A exhibited higher specific activity than AmyPG2, while T262A and R302A showed lower activity. The specific activity of mutant S231A was comparable to that of AmyPG2 (Table 2).

Table 2.

Biochemical characterization of the wild-type AmyPG2 and its mutants

Enzyme	Opt. temperature (°C)	Opt. pH	Opt. NaCl (M)	Specific activity without NaCl (U/mg)	Relative activity without NaCl (%)	Specific activity added 2.0 M NaCl (U/mg)	Relative activity added 2.0 M NaCl (%)
AmyPG2	25	8.0	2	4.2 ± 0.08	100	484.3 ± 12.3	100
S231A	25	8.0	2	4.3 ± 0.17	102	469.9 ± 16.4	97.0
I236A	25	8.0	2	6.4 ± 0.23	152	547.2 ± 18.8	113
I236V	25	8.0	2	9.7 ± 0.31	231	897.1 ± 23.6	185
T262A	30	8.0	2	0.73 ± 0.04	17.3	95.6 ± 4.1	19.7
S263A	25	7.5	2	5.9 ± 0.25	140	592.8 ± 20.5	122
R302A	25	7.5	2	1.4 ± 0.04	33.3	178.9 ± 6.9	36.9

Table 3 shows the comparison of kinetic parameters of AmyPG2 and three mutants with increased specific activity. These data of catalytic efficiency (k_cat/K_m) on the whole were consistent with the results of specific activity. Compared with AmyPG2, the mutant I236V exhibited the highest improvement in catalytic efficiency, with 2.31- and 2.51-fold increases with and without NaCl, respectively. Therefore, I236V was chosen for further measurements with the wild-type AmyPG2.

Table 3.

Kinetic parameters of the wild-type AmyPG2 and its mutants

Enzyme	Kcat without NaCl (/s)	Km without NaCl (mg/mL)	kcat/Km without NaCl (/s/mg/mL) (%)	Kcat added 2.0 M NaCl (/s)	Km added 2.0 M NaCl (mg/mL)	kcat/Km added 2.0 M NaCl (/s/mg/mL) (%)
AmyPG2	114.6	31.5 ± 1.2	3.6 (100)a	1247.5	5.8 ± 0.19	215.1 (100)a
I236A	173.1	29.2 ± 1.1	5.9 (164)b	1912.7	5.0 ± 0.24	379.3 (176)c
I236V	188.8	22.7 ± 0.9	8.3 (231)c	1899.4	3.5 ± 0.11	540.5 (251)d
S263A	161.7	26.9 ± 1.0	6.0 (167)b	1401.5	4.2 ± 0.15	333.9 (155)b

The same lowercase letters indicate no significant difference between groups (p < 0.05)

Potential application of AmyPG2 and I236V in the low-temperature hydrolysis of starch

The application potential of AmyPG2 and I236V for low-temperature starch hydrolysis was evaluated. The hydrolysis kinetics of 16 h at 25 °C were evaluated and compared with those of the commercial α-amylase PPA (Fig. 5). When 8% gelatinized mung starch was used as the substrate, both AmyPG2 and I236V exhibited high hydrolysis efficiency, maintaining such high efficiency for approximately 12 h. Prolonged incubation to 16 h resulted in only a slight increase in reducing sugars. The final reducing sugar yield of AmyPG was 512.5 ± 17.8 mg/g starch (equivalent to 51.2 ± 1.8% hydrolysis rate), while the final reducing sugar yield of I236V was 625.3 ± 23.8 mg/g starch (equivalent to 62.5 ± 2.4% hydrolysis rate), with a 1.22-fold enhancement. Under the same temperature and time (25 °C and 16 h), the final hydrolysis rate of PPA was 31.9 ± 1.9% (Fig. 5a). The hydrolysis efficiency of AmyPG2 and I236V towards gelatinized wheat starch (8%) was slightly lower. The final reducing sugar yield reached 392.4 ± 11.8 mg/g starch (equivalent to 39.2 ± 1.2% hydrolysis rate) for AmyPG2, versus 452.1 ± 9.3 mg/g starch (equivalent to 45.2 ± 0.9% hydrolysis rate) for I236V. PPA displayed a hydrolysis rate of 37.1 ± 1.5% (Fig. 5b). The results demonstrated the high efficiency of AmyPG2 and I236V in starch hydrolysis at low temperature, which could be applied to design new starch biorefinery processes.

Fig. 5.

Time courses of the hydrolysis of 8% gelatinized mung starch (a) and wheat starch (b) by AmyP2 (black square), the mutant I236V (green up-pointing triangle), and PPA (blue circle). The results were expressed as mean ± standard deviation (SD)

Potential application of AmyPG2 and I236V in the detergent industry

Compatibility with commercial laundry detergents

To assess the potential application of the two enzymes in laundry detergents, their stability in commercial detergents was initially assessed. The residual activities of AmyPG2 and I236V after incubation with seven different detergents at 25 °C for 1 h are shown in Fig. 6. Both enzymes showed good stability, retaining more than 55% of activity even in the harshest detergent (Chaoneng^®). Their performance was very similar, and the highest compatibility was observed in Blue Moon^®, yielding residual activities of 94.8% for AmyPG2 and 95.3% for I236V.

Fig. 6.

Stability of AmyP2 (black) and the mutant I236V (green) in different commercial laundry detergents. The results were expressed as mean ± standard deviation (SD)

Wash performance analysis

Chocolate- and tomato sauce-stained cotton textiles were used to evaluate the laundry performance of AmyPG2 and I236V. The enzyme was added to the detergent Blue Moon^® as an additive, where it had the highest stability. As shown in Fig. 7, α-amylase AmyPG2 significantly improved the detergent washing performance when compared to the control without enzyme. The stain removal ability of I236V was better than that of AmyPG2, especially for the chocolate stain. The results indicated that both AmyPG2 and I236V are promising candidates for use as laundry detergent additives.

Fig. 7.

Wash performance analysis of AmyP2 and the mutant I236V against cotton fabrics soiled with chocolate and tomato sauce. Lane T: cotton fabrics stained with tomato sauce; Lane C: cotton fabrics stained with chocolate. Control: stained cotton fabrics before wash; W: washed with distilled water containing NaCl; W + D: washed with distilled water containing NaCl and detergent Blue Moon^®; W + D + E1: washed with distilled water containing NaCl, detergent Blue Moon^®, and AmyP2; W + D + E2: washed with distilled water containing NaCl, detergent Blue Moon^®, and I236V

Discussion

AmyPG2 was a cold-adapted and alkalophilic α-amylase with the highest activity at 25 °C and pH 8.0 from the marine bacterium P. gaetbulicola. Comparison of AmyPG2 and seven characterized GH13_27 α-amylases showed that the α-amylases from Alteromonas macleodii B7 (Han et al. 2014), Catenovulum sp. X3 (Wu et al. 2017), and Pseudomonas sp. KFCC-10818 (Lee et al. 2008) are cold-adapted; however, their optimal temperature was higher than that of AmyPG2, ranging from 35 to 50 °C. Likewise, these enzymes, along with AmyPG2, are alkalophilic, exhibiting optimal pH values between 8.0 and 9.0. It is noteworthy that these cold-adapted and alkalophilic α-amylases within the GH13_27 subfamily are derived from marine bacteria, suggesting that marine-derived microorganisms are valuable resources for discovering novel amylases.

A dominant feature of the majority of cold-adapted enzymes is reduced thermal stability, which poses a major challenge to their industrial applications. Cold-active α-amylases from Pontibacillus sp. ZY, Zunongwangia profunda, and Pseudoalteromonas sp. 2–3 showed that the half-lives at 30–43 °C were only about 10–25 min (Table 4) (Fang et al. 2019; Qin et al. 2014; Sanchez et al. 2019). AmyPG2 exhibited 50% activity after 1.6 and 1.0 h at 35 and 40 °C, respectively, indicating better thermal stability compared to many other cold-adapted α-amylases.

Table 4.

Comparison of AmyPG2 with other characterized cold-adaptive amylases

Enzyme	Sources	Topt. (°C)	pHopt	Temperature stability	NaClopt (M)	Commercial detergent	Specific activity (U/mg)	References
Amy175	Pseudoalteromonas sp. M175	25	8.0	88.6% at 30 °C for 1 h	1.0	Tide®, Chaoneng®, Liby®, OMO®, and Blue Moon®	337.9	Wang et al. (2018)
PersiAmy1	Metagenomic library	10	9.0	61% at 90 °C for 3 h	Halotolerance	Softlan®, Persil® and Homecare®	4062.9	Ariaeenejad et al. (2021)
I3C6	Metagenomic library	30	8.0	80% at 35 °C for 1 h	Inhibitory effect	Green Balance® and Bio-tex®	5	Vester et al. (2015), Bendtsen et al. (2025)
/	Bacillus cereus GA6	22	9.0	nd	nd	Tide® and Ghari®	175.9	Kuddus (2013)
AmyZ	Zunongwangia profunda	35	7.0	50% at 35 °C for 25 min	1.5	nd	270.6	Qin et al. (2014)
AmyD-1	Bacillus sp. dsh19-1	20	6.0	50% at 40 °C for 1 h	2.0	nd	10.7	Dou et al. (2018)
AMI	Pseudoalteromonas sp. 2–3	20	8.0	63% at 40 °C for 10 min	nd	nd	36.7	Sanchez et al. (2019)
/	Cladophora hutchinsiae	10	6.0	63% at 10 °C for 14 days	nd	nd	89.7	Cakmak et al. (2022)
Rho13	Metagenomic library	35	8.0	nd	Inhibitory effect	nd	≈ 150	Bendtsen et al. (2025)
Ika2	Metagenomic library	30	6.0	nd	Inhibitory effect	nd	≈ 6	Bendtsen et al. (2025)
AmyZ1	Pontibacillus sp. ZY	35	7.0	50% at 30 °C for 12 min	nd	nd	≈ 12,621	Fang et al. (2019)
AmyPG2	P. gaetbulicola Gung47	25	8.0	50% at 25 °C for 5 h	2.0	Blue Moon®, Walch®, Diaopai®, Liby® and OMO®	4.2	This study

Halophilic α-amylases are inactive without NaCl (Wang et al. 2019; Fukushima et al. 2005). In contrast, halotolerant enzymes, such as α-amylases from Ulkenia sp. AH-2 (Shirodkar et al. 2020) and Nesterenkonia sp. F (Solat and Shafiei 2021), are active without NaCl, and NaCl can increase their enzymatic activities by two- and threefold, respectively. AmyPG2 showed activity under salt-free conditions, thus classifying it as a halotolerant α-amylase. Its specific activity was significantly increased by 115-fold upon the addition of NaCl. This degree of enhancement is greater than that of any known halotolerant α-amylase.

The kinetics of AmyPG2 and its mutants were analyzed using soluble starch as a substrate in the absence and presence of NaCl (Table 3). Among the mutants, I236V exhibited the lowest K_m regardless of the presence or absence of NaCl (p < 0.05). A decrease in K_m indicates increased substrate affinity, which may be due to the mutation from Ile to Val, shortening the side chain and reducing steric hindrance in the substrate-binding pocket (Fukunaga et al. 2004; Chen et al. 2025). Consistent with its reduced K_m, I236V exhibited the greatest improvement in catalytic efficiency (k_cat/K_m) among all mutants (p < 0.05).

By combining molecular docking with sequence logo analysis, we designed site-directed mutations of AmyPG2. Under optimal conditions, the I236V mutant was found to display a more than twofold enhancement in catalytic activity and exhibited specific activities as high as 3480.0 ± 57.2 and 2199.5 ± 33.8 U/mg toward mung starch and wheat starch, respectively. When the hydrolysis was performed at 25 °C, the final hydrolysis rates of I236V for 8% gelatinized mung and wheat starch were 62.5 and 45.2%, respectively, which are higher than those of typical medium-temperature PPA. There are few records on the cold hydrolysis of starch by cold-adapted α-amylases at temperatures of 25 °C and below. Thus, I236V is not only a mutant with enhanced hydrolytic activity but also a potential candidate enzyme for starch-saccharifying application.

Another important application of α-amylases is as additives in detergents. For cleaning textiles such as wool and silk, it is preferable to employ cold water to prevent fabric damage. Therefore, an ideal α-amylase for these detergent applications should combine several important properties: cold-active, alkaline, detergent-stable, and efficiently hydrolytic. Most α-amylases applied in laundry detergents do not exhibit these properties at the same time, operating optimally at 37–60 °C (Gupta et al. 2024; Suthar et al. 2024; Khelil et al. 2022). Only three cold-active α-amylases have been reported to maintain high stability in commercial detergents at the low temperatures (10–25 °C) (Table 4). Among these, the α-amylases from Pseudoalteromonas sp. M175 (Wang et al. 2018), B. cereus GA6 (Kuddus 2013), and a metagenomic library (Ariaeenejad et al. 2021) were further confirmed to effectively remove stains at low temperatures. Both AmyPG2 and its mutant I236V showed good stability with commercial detergents, particularly Blue Moon^®. Moreover, they demonstrated effective washing performance at 25 °C, thus making them suitable for use in the development of low-temperature detergents.

In conclusion, a novel α-amylase, AmyPG2, from P. gaetbulicola, was recombinantly expressed and purified. AmyPG2 possesses several remarkable characteristics: alkaline, cold-adapted, thermally stable, halotolerant, and detergent-stable. AmyPG2 and its enhanced mutant I236V thus represent valuable new candidates to meet the growing demand for industrial enzymes in low-temperature starch hydrolysis and detergent formulations.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

J. L.: Data analysis, Writing-Original, Experiment. J.Z. H.: Investigation, Methodology, Data analysis. L. X., X.R. X.: Experiment. Y.Z. X.: Resources, Visualization, Data Curation. Y. G.: Software, Validation. H. P.: Conceptualization, Resources, Supervision, Project administration.

Funding

This work was supported by the Natural Science Foundation of China (32170137).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files. The materials that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethical approval

Not applicable.

Competing interests

The authors declare no competing interests.