The present study does handle the cloning, heterologous expression, biochemical characterization, and in silico structural modeling of pullulanase type I from Metabacillus indicus for the first time ever. The molecular structure of Pull_Met was similar to the structure of other pullulanases homologues from other species such as pullulanases type I with the PDB entries: 2WAN, 2WDJ, 2E8Z, and 2YDT from Bacillus acidopullulyticus, Anoxybacillus sp. LM18-11, B. subtilis str.168, and S. pnumoniae, respectively. The aforementioned homologues pullulanases and Pull_Met molecular structures did share a uniform architecture with multiple domains: N-domain, CBM (carbohydrate binding module), catalytic domain, and C-domain (Xu et al. 2021). These CBM (e.g., CBM41, CBM48, and CBM68) frequently reported in the architecture of glycosidic hydrolases of family GH13, play a crucial role in the binding of pullulanases to the starch/glycogen and help facilitate the hydrolysis process (Janeček et al. 2017). At most, pullulanases type I CBM (s) like CBM41 and CBM48 are disordered with several modules of unveiled functions termed X modules. For instance, pullulanase type I of the PDB entry 2WAN had CBM 41 and CBM48 disordered with X-modules namely X25 and X45 of unknown functions. However, for Pull_Met and its structurally similar PDB entry 2E9B of B. subtilis str. 168 pullulanase type I, the architecture is much simpler as only X25 module of 104 residues preceding the CBM48 (Turkenburg et al. 2009).

The conserved motif of seven amino acids YNWGYNP was reported to predominate in all pullulanases pullulanase type I that only could attack α-1,6 glycosidic bonds of pullulan (Erden-Karaoğlan et al., 2019, Iqrar et al. 2020, Prongjit et al. 2022), this region would appear to be involved in substrate binding or catalytic activity (Bertoldo et al. 1999, Yamashita et al., 1997). Reportedly, this conserved motif does not exist in pullulanases type II that could attack both α-1,4 glycosidic bonds and α-1,6 glycosidic bonds in pullulan.

Likewise other pullulanases type I, belonging to GH13, Pull_Met had four conserved regions namely I, II, III, and IV localized in the catalytic cleft for substrate binding as shown in Fig. 6. The catalytic triad residues Asp410, Glu439, and Asp523 of Pull_Met were located on β-strand, β-strand, and loop in the β/α barrel, respectively. Whilst, the catalytic triad (Asp, Glu, and Asp) of other pullulanases type I (e.g., 2FH8, 2YA1, 2WAN, 6JEQ, 3WDJ, and 2E8Z) were reported to be localized all on β-strands (Xu et al. 2021). Reportedly, in GH13 pullulanases, the β-4-Asp, β-5-Glu, and β-7-Asp represented the catalytic nucleophile, proton donor, transition-state stabilizer, respectively.

The Pull_Met amino acid sequence lacked a signal peptide as deduced from Signal IP 6.0 server. Similarly, the amino acid sequences of pullulanases type I such as 2FH8, 2YA1, 2WAN, 6JEQ, 3WDJ, and 2E8Z did lack signal peptide sequence as revealed from the analysis on Signal IP 6.0 server. Conversely, pullulanase type I of Fervidobacterium pennavorans Ven5 showed a signal peptide of 28 amino acid residues (Bertoldo et al. 1999). As a rule of thumb, the signal peptide plays an essential role in protein secretion outside the cell. Nevertheless, the heterologous expression of majority of reported pullulanases type I have not realized high expression levels and effective extracellular secretion despite the presence of signal peptide sequence or secretory proteins as fusion partners (Albertson et al., 1997, Michaelis et al. 1985, Takizawa et al. 1985, Tomiyasu et al., 2001). Conversely, the pullulanase type I from Klebsiella pnumoniae 342, K. variicola AT-22, and K. variicola SHN-1 showed a signal peptide (Chen et al., 2013).

The molecular mass of Pull_Met (79.1 kDa) was localized in the range of reported molecular masses of pullulanases type I (70–140 kDa). For instance, the molecular masses of pullulanases type I from Anaerobranca gottschalkii (Bertoldo et al. 2004), F. pennavorans Ven 5 (Hii et al. 2012), Clostridium thermohydrosulfuricum (Saha et al. 1988),, Bacillus sp. S-1 (Lee et al. 1997), Thermatoga maritima (Kriegshäuser et al., 2000), and Bacillus naganoensis (Zhang et al., 2013) were 96, 77, 136.5, 80, 140,89, and 100 kDa, respectively.

Regarding the multimerization status (quaternary structure) of Pull_Met, the experimental data derived from native –PAGE (monomeric subunit) was in a good accordance with that of predicted data derived from SWISS-MODEL. Moreover, the quaternary structure of Pull_Met was in a good agreement with that of the majority of previously reported pullulanases type I (e.g., Exiguobacterium acetylicum (Qiao et al., 2015) and L amylophillus GV6 ) (Dakhmouche Djekrif et al. 2021), with the exception of pullulanases from F. pennivorans (Bertoldo et al. 1999) and Geobacillus thermoleovorans US105 (Zouari Ayadi et al. 2008), which have a dimeric structure.

Generally speaking, highlighting the enzyme’s physico-biochemical properties is considered a crucial detrimental factor in the booklet of upcoming industrialization stage for an enzyme’s ultimate and effectual exploitation. The optimal temperature of Pull_Met is at 40 oC which indicates that it is cold-adapted pullulanase type I. The literature of review has a few reports considering cold-adapted pullulanase type I with an optimal temperature between 35 and 50 oC. For instance, pullulanases type I from Paenibacillus polymyxa Nws-pp2 (Wei et al. 2015), Shewanella arctica (Elleuche et al. 2015), Exiguobacterium sp. SH3 (Rajaei et al. 2015)d methanolicus PB1 (Zheng et al. 2021) exhibited an optimal temperature at 35, 35, 45, and 50 oC, respectively. Likewise Pull_Met, pullulanase type I from a hot-spring metagenome (Thakur et al. 2021), B. subtilis BK07 (Erden-Karaoğlan et al., 2019), and B. subtilis PY22 (Erden-Karaoğlan et al., 2019) and Priestia koreensis HL12 (Prongjit et al. 2022) showed an optimal temperature at 40 oC. Conversely, there is a plethora of reports addressing thermostable pullulanase type I with an optimal temperature above 50 oC. The optimal temperature of thermostable pullulanases type I from F. pennavorans Ven5 (Koch et al. 1997), Anaerobranca gottschalkii (Bertoldo et al. 2004), Bacillus thermoleovorans US105 (Messaoud et al. 2002), Thermotoga maritima (Kriegshäuser et al., 2000) showed an optimal temperature at 65, 70, 75, and 90 oC, respectively. Pull_Met, a cold-adapted pullulanase type I, had a lower optimal temperature (40 oC) than other described cold-adapted pullulanases (45–50 oC) (e.g., pul-SH3 and pulPB1). As a result, Pull_Met’s advantages would be utilized in bioprocesses carried out at moderate temperatures. Currently, the usage of cold-adapted pullulanase type I in industry is much more preferable to the usage of thermostable homologues pullulanase type I from the standpoint of cost-effectiveness and energy saving.

The half-life time of the enzyme is another issue that would put constrains concerning the likely applied temperature in the bioprocesses. Enzymes with long -half life time is much preferable to homologues enzymes with short half life time in pro-longed bioprocesses. Hence, longevity in bioprocesses is another issue that would determine the choice of an enzyme with a pro-longed half life time. In this context, the Pull_Met exhibited typical characteristics of cold-adapted pullulanase type I including low-thermostability at elevated temperatures maintaining around 50 and 30% of its activity at 15–30 oC after 2 h and at 45 oC after 30 min, respectively. However, pullulanase type I of Shewanella arctica (Elleuche et al. 2015) retained 76% of its activity at 30 oC after 2 h. For cold-adapted pullulanase type I of Paenibacillus polymyxa Nws-pp2 (Wei et al. 2015), the retained activity after 2 h was 70 and 60% after 300 min at 35 and 40 oC, respectively. While, the cold-adapted pullulanase type I of Exiguobacterium sp. SH3 (Rajaei et al. 2015) displayed 100% retained activity after 60 min at 40 oC.

The thermostability issue of a given globular protein is reportedly to be linked with the aliphatic -index which is defined as the relative volume of the protein engaged with aliphatic side chains (i.e., alanine, isoleucine, valine, and leucine). Reportedly, the aliphatic index of protein from thermophilic bacteria is higher than the ordinary proteins (Ikai 1980). The aliphatic index of the thermostable pullulanases type I from thermophilic bacteria such as Fervidobacterium pennivorans, Hymenobacter mucosus, and Desulfurococcus mucosus exhibited higher aliphatic indices of 86.12, 86.6, and 90, respectively compared to that of Pull_Met of 81.6. Conversely, the aliphatic indices of the thermostable Geobacillus thermoleovorans, Thermococcus hydrothermalis, and Thermoanaerobacter thermohydrosulfuricus were of 81.5, 81.5, and 74.42, respectively which were lower than that of Pull_Met. This would in turn reflect that the thermostability issue of a given protein is correlated with factors other than the aliphatic index. The index may be regarded as a positive factor for the increase of thermostability of globular proteins.

Concerning the optimal pH for previously reported pullulanases type I, the optimal pH spanned from 4.5 to 8.5. In this context, the Pull_Met displayed an optimal pH of 6.0 which was well- compatible with the reported range of the optimal pH for pullulanases type I. Meanwhile, pullulanase type I from Lactococcus lactis (Waśko et al., 2011), Bacillus methanolicus PB1 (Zhang et al. 2020), Priestia koreensis HL12 (Prongjit et al. 2022), Bacillus megaterium Y103 (Wu et al. 2022), and Anaerobranca gottschalkii (Bertoldo et al. 2004), exhibited an optimal pH of 4.5, 5.5, 6.0, 6.5, and 8, respectively. The slight acidic to near neutral pH optima for Pull_Met could be explained by the perception that the enzyme is most likely secreted internally in the cytoplasm, which displays a low pH compared to the pH in the external environment (Krulwich et al. 1997). This cellular localization of Pull_Met was additionally evidenced by the absence of a signal peptide in Pull_Met amino acid sequence as predicted by Signal IP 6.0.

The pH stability of an enzyme is an issue of a paramount importance in enzymes- dependent bioprocesses. An enzyme wide a broad range of pH stability would be more preferable than its homologues to cope well under harsh conditions. In the light of this conception, the Pull_Met showed a full stability for 5 h under a wide range of pH(s) from 2.6 to 10.0 except pH(s) 8.0 and 8.5. The pullulanase type I from Exiguobacterium acetylicum YH5 (Qiao et al., 2015) exhibited less stability (93%), over a wide range of pH 4–10 within 30 min, compared to that of Pull_Met. Whilst, pullulanase type I of Paenibacillus barengoltzii (Liu et al. 2016) demonstrated pH stability (80–100%) over a wide range of pH(s) from 5.0 to 10.5 after 30 min. A narrow-range of pH stability (6.0-8.5) as displayed by pullulanase type I from B. megaterium Y103 (Wu et al. 2022) with a retained activity of over 80% after 30 min.

The aptness of Pull_Met for working effectively in slightly acidic to near neutral settings in bioprocesses would be controlled mostly by its pH stability. The discrepancy in optimal pH and temperature for pullulanases type I from thermophilic, mesophilic, and psychrotolerant bacteria might be attributed to the strain difference, nature of the habitat of these bacteria, where the latter would in turn impose the mechanisms of acclimatization under harsh conditions in extremophiles habitats in terms of whole proteome with unique properties for each strain.

Reportedly, pullulanases type I of bacterial origin show metal ion preference that would promote their activity. Metal ion preference by pullulanases type I varied widely among different strains. The Pull_Met activity was not stimulated by Ca2+. Similar phenomenon was traced in the pullulanases from Bacillus acidopullulyticus (Stefanova et al. 1999) and Anoxybacillus sp. LM18-11(Hii et al. 2012) Despite, searching the NCBI conserved domain database (CDD) revealed that pullulanase (PbPulA) had three Ca2+ binding sites at D216, E224, and E245. Moreover, pullulanases type I from Bacillus sp. CICIM 263 (Stefanova et al. 1999) and Thermococcus hydrothermalis (Gantelet et al. 1998) Vent had not only been activated by Ca2+ but also showed enhanced thermostability. Likewise Pull_Met, the P. polymyxa Nws-pp2 (Wei et al. 2015) and Thermus caldophilus GK-24 pullulanases type I (Kim et al. 1996) were significantly stimulated by Mn2+, meanwhile Cu2+ exerted significant dramatic drop in enzyme activity. The pullulanase type I from E. acetylicum YH5 (Qiao et al., 2015) was significantly enhanced by Mn2+ and Fe2+ but was significantly inhibited by Cu2+ after 30 min pre-incubation. Likewise Pull_Met, pullulanase type I from Lactococcus lactis IBB 500 (Waśko et al., 2011) showed complete loss in enzyme activity after 30 min pre-incubation with Hg2+. Unlike Pull_Met, the pullulanase type I from Thermus caldophilus GK-24 (Kim et al. 1996) was stimulated significantly in presence of Ni2+.

Analysis of pullulan end products is an important issue in the context of determining the type of Pull_Met. The end products of pullulan hydrolysis by Pull_Met indicated its affiliation to group type I as long as the maltotriose was the sole end products of hydrolysis. This sole end product of pullulan hydrolysis along with the inability to attack α-amylose would confirm that Pull_Met was capable of attacking α,1–6 glycosidic linkage not α-1,4 glycosidic linkage. Additionally, the in silico sequence analysis of Pull_Met, indicating its affiliation to pullulanases type I, was in a complete agreement with the experimental results of pullulan hydrolysis end products. Likewise Pull_Met, pullulanases type I from B. naganoensis (Zhang et al., 2013), Exiguobacterium sp. SH3 (Rajaei et al. 2015) Exiguobacterium acetylicum YH5 (Qiao et al., 2015), Geobacillus subterraneus strain KCTC 3922 (Chen et al. 2022) showed only maltotriose as the end products of pullulan hydrolysis. The main end product of pullulan hydrolysis by Pull_Met, maltotriose, would reflect its likely exploitation in starch processing to produce maltotriose with a high degree of purity. Reportedly, pullulanases type I showed broad range of substrate specificity especially on polysaccharides substrates with α-1,6 glycosidic linkages such as pullulan, glycogen, amylopectin, and starch. In this context, Pull_Met obeyed this rule as it showed an activity on starch and dextrin while the ultimate activity was evidenced on pullulan.

The EDTA exhibited full dramatic loss in Pull_Met activity that would reflect that Pull_Met was a metallo-enzyme (i.e., presence of metal ions was essential for enzyme activity). Likewise Pull_Met, pullulanases type I from E. acetylicum YH5 (Qiao et al., 2015) and Geobacillus kaustophilus DSM7263 (Li et al. 2018) were metalloenzymes.

The activity of Pull_Met was stimulated by the reducing agents, β-mercaptoethanol at 10 mM. Similar results were obtained in the previous studies on pullulanases type I (Thakur et al. 2021; Wei et al. 2015). It is likely that β-mercaptoethanol would inhibit the oligomerization of the enzyme (leading to its inactivation) by breaking the disulfide bonds between monomeric subunits of the enzyme.

Reportedly, detergents imposed a great effect on stability and activity of amylolytic enzymes including pullulanases (Thakur et al. 2021). The presence of (0.1, 0.25, and 0.5 mM) of non-ionic detergents (Tween-80, Twen-20, and Triton X-100) imposed a significant enhancement in the Pull_Met activity (up to 151%) after 30 min pre-incubation (Fig. 3A) This was in accordance with previous reports describing the role of non-ionic detergents on pullulanase type I (Elleuche et al. 2015; Wu et al. 2022).

Regarding SDS, the retained Pull_Met activity was 47.11 ± 7.09 and 38.9% ± 2.02, after 30 min pre-incubation with SDS at 0.25 and 0.5 mM, respectively. In this regard, the activity of pulA from Thermotoga neapolitana was reported to decline to 17% in presence of 35 mM SDS (Kang et al. 2011). Moreover, the pullulanases type I from F. pennavorans Ven5 (Bertoldo et al. 1999), G. thermoleovorans US105 (Zouari Ayadi et al. 2008)d arctica (Elleuche et al. 2015) were reported to be completely inhibited in presence of 1, 3.5, and 35 mM SDS, respectively. Unlike Pull_Met, Pul-SH3 did prove to be SDS tolerant and its activity was being traced up to 350 mM of SDS (Rajaei et al. 2015).

A significant dramatic decline in Pull _Met activity up to 22.36% was noticed after 30 min pre-incubation with CTAB. Similarly, the pullulanase type I activity of S. arctica was lowered by increasing the concentration of CTAB (Elleuche et al. 2015).

The tolerance against detergents is a significant characteristic by which enzymes are evaluated for their potential industrial application.

At most, amylupullulanases (pullulanase type II) did show detergents stability and several studies reported the washing performance of these enzymes (Dakhmouche Djekrif et al. 2021). Conversely, a few studies did address the likely potential of pullulanase type I in the detergent industry (confined only to Pul-SH3 (Rajaei et al. 2015) and PulY103 (Wu et al. 2022)). Interestingly, in the presence of commercially detergents, Pull_Met retained the majority of its activity (around 98%) after 30 min pre-incubation with Oxi TM and Tide ™. However, around 89% of activity was retained after 30 min pre-incubation with Persil ™, Ariel ™, and Art ™. Hence, the enzyme would display a great potential in the detergent industry as a detergent additive. Among the studies that evaluated the wash performance of pullulanases (Wu et al. 2022), where pullulanase type I from B. megaterium Y103 exhibited the maximal R (the value of detergency) and P (rate of the value of detergency) when combined with the commercial laundry detergent BlueMoon. The pullulanase type I from Exiguobacterium sp. SH3 (Rajaei et al. 2015) exhibited stability of 80.4 and 93.7% in the presence of two commercial detergents, Rika (7.5% v/v) and Fadisheh (2.5% w/v), respectively.

Most of reported studies on pullulanases did highlight the stability of pullulanases type II towards organic solvents (Siroosi et al. 2014). No previous studies reported the solvent stability of pullulanases type I. Consequently, the comparison of Pull_Met stability towards organic solvents would not be justified yet. Pull_Met was stable in presence of methanol, ethanol, and butanol.

Comparing the Km and Vmax values among different enzymes is not easy task since there is a wide discrepancy in the substrates and the reaction conditions. As a rule of thumb, a low Km value for a given enzyme does confer the high specificity of this enzyme toward the substrate and vice versa. The km value of pullulanase type I of microbial origin varied widely. Obviously, Pull_Met exhibited a Km of 2.369 mg/mL, which was much smaller than those of other pullulanases type I from a hot-spring metagenome with Km of 15.25 mg/mL (Thakur et al. 2021), G. subterraneus strain KCTC 3922 with Km of 4.37 mg/mL (Chen et al. 2022), B. polymyxa Nws-pp2 with Km of 4.0 mg/mL (Wei et al. 2015), and Priestia koreensis HL12 with Km of 3.81 mg/mL. This in turn would reflect the high specificity of Pull_Met on pullulan compared to the aforementioned pullulanases type I.

Interestingly, Pull_Met exhibited potential in raw ex potato starch saccharification in synergistic co-operative action with CA-AM21 as concluded from the liberated reducing sugars in terms of glucose from the raw starch.

Typically, raw substrate hydrolysis requires the synergistic interaction of enzyme composites to achieve complete saccharification (conversion yield ≥ 80%), such as cooperation of α-amylase and pullulanase in starch hydrolysis (Pan and Lee 2005; Prongjit et al. 2022).

This indicated that pull_Met is suitable to use as the main single amylolytic enzyme, combined with other degradation enzymes, to achieve complete saccharification and could be used to develop efficient starch saccharification and modification processes.

In both the academic and industrial sectors, the vast majority of type I pullulanase has only ever been assigned to mesophilic and thermophilic homologues. Nevertheless, cold-adapted pullulanase type I is a crucial class of enzymes in bioprocessing because one can stop a reaction at low temperatures without releasing undesirable end products due to unwanted side reactions in the food industry, which are typically caused by using pullulanase type I’s thermophilic and mesophilic homologues. Pull_Met is considered one paradigm of a little bit previously reported cold-adapted pullulanase type I in the academic sector so far. In conclusion, Pull_Met is a cold-adapted type I pullulanase with added value in two commercial sectors: saccharification and detergency, despite the dearth of cold-adapted type I pullulanase.