**Triinu Visnapuu, Aivar Meldre, Kristina Põšnograjeva, Katrin Viigand, Karin Ernits and Tiina Alamäe \***

Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia; triinu.visnapuu@ut.ee (T.V.); aivarmeldre@gmail.com (A.M.); kristina.poshnograjeva@gmail.com (K.P.); katrin.viigand@gmail.com (K.V.); karin.ernits@gmail.com (K.E.) **\*** Correspondence: tiina@alamae.eu

Received: 28 November 2019; Accepted: 30 December 2019; Published: 31 December 2019

**Abstract:** Genome of an early-diverged yeast *Blastobotrys* (*Arxula*) *adeninivorans* (*Ba*) encodes 88 glycoside hydrolases (GHs) including two α-glucosidases of GH13 family. One of those, the *rna\_ARAD1D20130g*-encoded protein (*Ba*AG2; 581 aa) was overexpressed in *Escherichia coli*, purified and characterized. We showed that maltose, other maltose-like substrates (maltulose, turanose, maltotriose, melezitose, malto-oligosaccharides of DP 4-7) and sucrose were hydrolyzed by *Ba*AG2, whereas isomaltose and isomaltose-like substrates (palatinose, α-methylglucoside) were not, confirming that *Ba*AG2 is a maltase. *Ba*AG2 was competitively inhibited by a diabetes drug acarbose (Ki = 0.8 μM) and Tris (Ki = 70.5 μM). *Ba*AG2 was competitively inhibited also by isomaltose-like sugars and a hydrolysis product—glucose. At high maltose concentrations, *Ba*AG2 exhibited transglycosylating ability producing potentially prebiotic di- and trisaccharides. Atypically for yeast maltases, a low but clearly recordable exo-hydrolytic activity on amylose, amylopectin and glycogen was detected. *Saccharomyces cerevisiae* maltase MAL62, studied for comparison, had only minimal ability to hydrolyze these polymers, and its transglycosylating activity was about three times lower compared to *Ba*AG2. Sequence identity of *Ba*AG2 with other maltases was only moderate being the highest (51%) with the maltase MalT of *Aspergillus oryzae*.

**Keywords:** *Arxula adeninivorans*; glycoside hydrolase; α-glucosidase; maltose; panose; amylopectin; glycogen; inhibition by Tris; transglycosylation

#### **1. Introduction**

A non-conventional yeast *Blastobotrys adeninivorans* (syn. *Arxula adeninivorans*) belongs to a basal clade of Saccharomycotina subphylum and diverged in the evolution of fungi long before *Saccharomyces* [1–5]. A recent study states that the divergence of basal Saccharomycotina from *Saccharomyces cerevisiae* took place between 200 and 400 million years ago [4].

*B. adeninivorans* has several biotechnologically relevant properties: accumulation of lipids [6], salt tolerance, temperature-induced filamentation that promotes protein secretion and the ability to use a wide range of carbon and nitrogen sources, including purines, tannin and butanol, that are unusual nutrients for yeasts [2,7]. *B. adeninivorans* has been engineered for butanol production, applied in kits for the detection of hormones and dioxine in water as well as for manufacturing of tannase and cutinases [7]. Some other enzymes of *B. adeninivorans* have also been investigated, including alcohol dehydrogenase [8], extracellular glucoamylase [9] and invertase [10]. A highly active endo-inulinase of *B. adeninivorans* was cloned and recently characterized [11]. The genome of *B. adeninivorans* was sequenced in 2014 [2].

The genes potentially encoding α-glucosidases in the genomes of non-conventional yeasts were analysed in Viigand et al. [12]. The genes encoding two putative α-glucosidases designated as AG1 and AG2 were revealed in genome of *B. adeninivorans.* In the genomes of most yeasts addressed in Viigand et al. [12], the α-glucosidase genes resided in maltose utilization (*MAL*) clusters, whereas no *MAL* clusters were detected in *B. adeninivorans*.

α-Glucosidases have been popular objects to study protein evolution and phylogenesis [13–16], but they also have a biotechnological value. Thus, some of them have a high transglycosylating activity thanks to which they produce prebiotic oligosaccharides and potential functional food ingredients such as panose, melezitose, isomelezitose and isomalto-oligosaccharides [17–21]. For example, the α-glucosidase of *S. cerevisiae* produced isomelezitose from sucrose when the substrate concentration was high [21]. Transglycosylating ability of maltose by the α-glucosidase of *Xanthophyllomyces dendrorhous* (syn. *Pha*ffi*a rhodozyma*) has been studied in detail, and synthesis of tri- and tetrasaccharides with α-1,6 linkages was detected. This enzyme certainly has a biotechnological potential—it produced 3.6 times more transglycosylation products than the *S. cerevisiae* α-glucosidase studied at the same conditions [17,20].

Considering α-glucosidases of yeasts, they have mostly been studied in *S. cerevisiae* as these enzymes are crucial in baking and brewing [22]. *S. cerevisiae* has two types of α-glucosidases—maltases (EC 3.2.1.20) and isomaltases (EC 3.2.1.10)—that differ for substrate specificity. Maltases degrade maltose and maltose-like sugars, i.e., maltotriose, turanose and maltulose, but cannot degrade isomaltose and isomaltose-like sugars (α-1,6 linkages) such as palatinose. Both types of enzymes hydrolyze sucrose and a synthetic substrate *p*-nitrophenyl-α-d-glucopyranoside (*p*NPG) [15,16,23,24]. At the same time, some yeasts such as *Ogataea polymorpha* and *Sche*ff*ersomyces stipitis*, have promiscuous maltase-isomaltases that hydrolyze maltose- and isomaltose-like substrates [12,16,25].

In the current work, we expressed heterologously in *Escherichia coli* and biochemically characterized the α-glucosidase *Ba*AG2 of *B. adeninivorans* encoded by *rna\_ARAD1D20130g*. We confirmed that *Ba*AG2 is a maltase with a considerable transglycosylating activity. Not typical for yeast maltases, *Ba*AG2 had exo-hydrolytic activity on amylose, amylopectin and glycogen. *Ba*AG2 is the first maltase characterized from *B. adeninivorans.*

#### **2. Results**

#### *2.1. In Silico Analysis of BaAG2*

According to annotations provided at the MycoCosm website [26], the genome of *Blastobotrys* (*Arxula*) *adeninivorans* [2] encodes 185 carbohydrate-active enzymes, including 88 glycoside hydrolases (GHs) assigned to different families. When mining the genome of *B. adeninivorans* [2] for the genes related to maltose hydrolysis, we found two genes encoding intracellular GH13 family proteins. Respective proteins were designated as AG1 and AG2 [12]. In MycoCosm, the AG1 was annotated as a protein similar to maltase Mal1 of *Schizosaccharomyces pombe* and the AG2 as similar to maltases of filamentous fungi *Aspergillus* and *Penicillium*. Both of these proteins were predicted to lack a signal peptide and to locate intracellularly. We confirmed this by using the SignalP program (see Materials and Methods). Aside from these two GH13 proteins, three putative extracellular α-glucosidases of GH31 family were detected in *B. adeninivorans* genome (see Table S1 of Supplementary Materials). Table S1 also includes two *B. adeninivorans* enzymes that have been experimentally studied: a secreted invertase AINV belonging to GH31 family [10] and a secreted glucoamylase [9] of GH15 family.

Substrate specificity of α-glucosidases can be *in silico* predicted based on so-called amino acid signature—a set of amino acids that locate in the vicinity of the substrate-binding pocket [12,15,27,28]. The upper panel of Figure 1 shows the amino acid signature of *O. polymorpha* maltase-isomaltase MAL1, *S. cerevisiae* maltase MAL62, isomaltase IMA1, and *B. adeninivorans* AG2. The amino acids of these proteins corresponding to Val216 of *Sc*IMA1 are shown inside a red frame as this position is considered of key importance in selective substrate binding [28].


**Figure 1.** Amino acid signature of yeast α-glucosidases, including *B. adeninivorans* AG2 (upper panel) and their designation on the three-dimensional (3D) structure of *S. cerevisiae* isomaltase IMA1 in complex with isomaltose (RCSB Protein Data Bank, PDB: 3AXH [29]) (lower panel). Location of Val216 in the structure is marked with a red circle.

We then visualized the *S. cerevisiae* IMA1 structure in complex with isomaltose (PDB: 3AXH) [29] using PyMol [30] in order to display all these amino acids (Figure 1, lower panel). In *Ba*AG2, a Thr is present at position of Val216 and therefore we predicted that this enzyme is most probably a maltase. However, as maltase-isomaltases also have a Thr at that position (Figure 1, upper panel; [12,16]), based on the amino acid signature, *Ba*AG2 may also be a promiscuous enzyme with a wide substrate spectrum like *O. polymorpha* MAL1.

Figure 2 presents fragments of sequence comparison of *Ba*AG2 with those of five experimentally studied maltases from GH13 family: two from bacteria, two from yeasts and one from a filamentous fungus *Aspergillus*. The identity matrix of these proteins is presented in Supplementary Materials (Table S2). Though the proteins aligned sufficiently well over the entire sequence, *Ba*AG2 showed only moderate sequence identity with the other maltases ranging from 35% with *Halomonas* sp. H11 α-glucosidase to 51% with *Aspergillus oryzae* maltase MalT (Table S2). *In silico* assay of α-glucosidases of non-conventional yeasts [12] identified a putative α-glucosidase protein AG1 of *Lipomyces starkeyi* as the closest homologue (50% identity) of *Ba*AG2. The amino acid signature of the *Lipomyces* protein was YTVNKLSHE, and it was, therefore, predicted as a maltase [12].

The GH13 enzymes use an Asp (D) as a nucleophile and a Glu (E) as an acid-base catalyst [31]. Additionally, an Asp of the conserved 'NHD' motif serves as a transition state stabilizer [32]. In the *Ba*AG2 protein, Asp216 was predicted as a nucleophile, Glu274 as an acid-base catalyst and Asp348 as a stabilizer (Figure 2). Thr217 is located next to the catalytic nucleophile Asp216 in *Ba*AG2 (Figure 2). In maltases and maltase-isomaltases, either Thr or Ala is present at respective position, whereas in isomaltases, a Val is present [12,15,27,28], indicating that a Val residue at this position interferes with hydrolysis of maltose-like substrates. Indeed, if respective Thr was substituted with Val in *O. polymorpha* maltase-isomaltase, utilization of maltose-like sugars was severely hampered [16]. Furthermore, after substitution of Val216 in *S. cerevisiae* IMA1 with Thr, the isomaltase IMA1 gained the ability to hydrolyze maltose [28,29].


**Figure 2.** Fragments of sequence alignment of six maltases. *Ba*AG2, *Blastobotrys adeninivorans* AG2 (580 aa); *Sp*Mal1, *Schizosaccharomyces pombe* Mal1 (579 aa, NP\_595063.1) [33]; *Sc*MAL62, *Saccharomyces cerevisiae* MAL62 (584 aa, P07265) [23]; *Gs*AG, *Geobacillus stearothermophilus* exo-α-1,4-glucosidase (555 aa, BAA12704.1) [34]; *Ha*AG, *Halomonas* sp. H11 α-glucosidase (538 aa, BAL49684.1) [35]; *Ao*MalT, *Aspergillus oryzae* maltase MalT (574 aa, XP\_001825184.1) [36]. Highlights: catalytic nucleophile (turquoise), acid-base catalyst (green), a transition state stabilizer (yellow) and a residue crucial for substrate specificity (red). Cc, Clustal consensus. Marking below the sequence alignment is according to Clustal consensus showing conservation: \* positions with fully conserved residue; : positions with residues of strongly similar properties; . positions with residues of weakly similar properties.

#### *2.2. Maltose-Like and Isomaltose-Like Sugars Are Growth Substrates for B. adeninivorans*

According to the information present in the CBS-KNAW culture collection [37], *B. adeninivorans* CBS 8244 used in current work assimilates following α-glucosidic sugars: maltose, sucrose, melezitose, trehalose and α-MG. Of those, melezitose is a maltose-like sugar, and α-MG (a synthetic analogue of isomaltose [38]) is an isomaltose-like sugar. Glucose and many other monosaccharides are also assimilated. We asked if *B. adeninivorans* can also assimilate some other α-glucosidic sugars. We cultivated *B. adeninivorans* on Yeast Nitrogen Base (YNB) mineral medium containing 2 g/L of sugars indicated in Figure 3, and evaluated growth according to an optical density (OD) of 600 nm achieved by 24 h of growth (Figure 3). Our assay confirmed that five above-mentioned α-glucosidic sugars were indeed all assimilated. In addition, maltotriose, maltulose, turanose (maltose-like sugars) as well as isomaltose and palatinose (an isomaltose-like sugar) were identified as new growth substrates for this yeast. Thus, *B. adeninivorans* grows on both maltose-like and isomaltose-like sugars, meaning that it should possess enzymes for the hydrolysis of both types of sugars.

**Figure 3.** Growth of *B. adeninivorans* on sugars (supplemented at 2 g/L) evaluated by an optical density (OD) of the culture at 600 nm achieved by 24-h cultivation on a microplate at 37 ◦C. Isomaltose and isomaltose-like sugars are indicated by dark blue bars. Error bars are representing standard deviations (SD) of two independent experiments with two replicates.

#### *2.3. Cloning of the* Ba*AG2 Gene and Heterologous Expression of the* Ba*AG2 Enzyme*

The *Ba*AG2 protein deduced from the gene is 580 aa long. The protein was predicted as intracellular—no secretion signal was detected by the SignalP program v. 5.0 [39]. *Ba*AG2 was overexpressed in *E. coli* with the His6-tag in its C-terminus that enabled purification of the protein using Ni2+-affinity chromatography. The calculated molecular weight of the protein was 67.9 kDa and a prominent band of respective size was detected by electrophoresis of the lysate produced from induced *E. coli* cells overexpressing the *AG2* gene (Figure S1). The *E. coli* lysate exhibited catalytic activity of 1 mM *p*NPG hydrolysis at 30 ◦C (71 U/mg), which after purification of the protein increased 5.8 times, reaching 411.5 U/mg.

#### *2.4. Properties of* Ba*AG2*

#### 2.4.1. Dependence of the *Ba*AG2 Activity on Temperature and pH. Thermal Stability of *Ba*AG2

The pH optimum of *Ba*AG2 was in moderately acidic region—from 5.5 to 6.5 (Figure S2). At pH 7.5, the activity was 81% from the maximum, and at pH 8.5, it was decreased to 52%. At pH 4.5 and 4.4, the respective values were 93 and 15%. Thus, the activity of *Ba*AG2 was significantly reduced at pH values below 4.5 and over 8.5 (Figure S2). The pH optimum of the *O. polymorpha* maltase is 6.0–6.5 [40], of *S. cerevisiae* maltase 6.5–6.8 [23,24] and of *Schizosaccharomyces pombe* maltase 6.0 [33]. In the current work, we routinely used the buffer with pH of 6.5 to characterize substrate specificity, kinetics and other properties of the enzyme.

As shown in Figure 4 (left panel), catalytic activity of *Ba*AG2 was the highest at 45 ◦C being 24% higher than activity measured at 30 ◦C—the temperature we routinely used for enzyme activity assay. Figure 4 shows that at temperature over 50 ◦C, the activity of the enzyme rapidly declined. Thermal stability assay of *Ba*AG2 indicated that the enzyme was rather thermolabile: if kept for 30 min at temperatures above 37 ◦C, its catalytic activity reduced significantly (Figure 4, right panel). Therefore, we routinely performed enzymatic assays at 30 ◦C since some reactions (e.g., transglycosylation and polysaccharide degradation assays) were conducted up to several days.

**Figure 4.** The effect of temperature on activity (left panel) and stability (right panel) of *Ba*AG2. For the thermostability assay, the enzyme was incubated for 30 min at the indicated temperature and residual activity was determined at 30 ◦C with *p*NPG as a substrate (see Materials and Methods, paragraph 4.4. for details). SDs of two to three replicates at each temperature are shown by error bars.
