Fructofuranosidase, in Rhizopus Microsporus

microorganisms

Article Identiﬁcation of sucA, Encoding β-Fructofuranosidase, in Rhizopus microsporus

Yoshitake Orikasa, Yuji Oda and Takuji Ohwada *

Department of Life and Food Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan; [email protected] (Y.Or.); [email protected] (Y.Od.) * Correspondence: [email protected]; Tel.: +81-155-49-5552

Received: 22 January 2018; Accepted: 13 March 2018; Published: 13 March 2018

Abstract: Rhizopus microsporus NBRC 32995 was found to hydrolyze fructooligosaccharides (FOS), as well as sucrose, almost completely into monosaccharides through the production of sufﬁcient amounts of organic acids, indicating that the complete hydrolysis of FOS was caused by the secretion of β-fructofuranosidase from fungal cells. Thus, the sucA gene, encoding a β-fructofuranosidase, was ampliﬁed by degenerate PCR, and its complete nucleotide sequence was determined. The total length of the sucA gene was 1590 bp, and the SucA protein of R. microsporus NBRC 32995 belonged to clade VIa, which also contains Rhizopus delemar and is closely related to Saccharomycotina, a subdivision of the Ascomycota.

Keywords: Rhizopus microsporus; β-fructofuranosidase; sucA gene; sucrose; fructooligosaccharides

1. Introduction Rhizopus fungi are widely distributed in the environment, including in soil and on plant surfaces, and are known as “kumonosukabi” in Japan. Some Rhizopus strains have been recognized as harmful, causing infectious disease in humans and plants [1]. However, Rhizopus strains have a strong ability to convert sugar into organic acids, which are widely used as raw materials and additives in foods, cosmetics, and medicines. For example, Rhizopus strains are used to produce fermented foods in Asia [2], such as the traditional Indonesian soybean product, tempeh [3]. Rhizopus oryzae is one of the organic acid-producing Rhizopus species, and is classified into two groups: the lactic acid-producing R. oryzae (LA group) and the malic-fumaric acid-producing Rhizopus delemar (FMA group). These classifications are supported by genetic and phylogenetic analyses [4,5]. Several types of microbial enzymes are known to be involved in the degradation of sugars such as sucrose and fructooligosaccharides (FOS), and widely distributed in plants, yeasts, and bacteria [6]. For example, β-fructofuranosidase (EC3.2.1.26) hydrolyzes sugars from fructosides, but glucoamylase (EC3.2.1.3) hydrolyzes sugars from glucosides. In particular, β-fructofuranosidase is useful for the production of organic acids owing to its stability even at low pH levels [7,8]. Thus, the distribution and classification of enzymes with β-fructofuranosidase activity have been previously investigated among several strains of R. oryzae and R. delemar. The results showed that the sucA gene encoding β-fructofuranosidase was present in only a few strains of R. delemar [7,8], and its DNA sequence revealed that the sucA genes in R. delemar strains belonged to a group of fungi distinct from other zygomycetes, but closely related to ascomycetes [8]. It was reported that the SucA (i.e., β-fructofuranosidase) can be classified into seven clades based on its conserved amino acid sequence [9]. The Rhizopus filamentous fungi are classified into three groups, Rhizopus stolonifer, R. oryzae (including R. delemar), and Rhizopus microsporus, based on culture temperature, the sporangium, and the size of the sporangiophore [5,10,11]. Thus, in this study, R. microsporus and R. stolonifer strains

Microorganisms 2018, 6, 26; doi:10.3390/microorganisms6010026 www.mdpi.com/journal/microorganisms Microorganisms 2018, 6, 26 2 of 11 were evaluated for the production of organic acids and ethanol as well as the distribution of the sucA gene. In particular, R. microsporus is known to exhibit an excellent ability to produce lactic acid and grow at high temperatures [12]. Therefore, the hydrolytic activity of the R. microsporus strain against both sucrose and fructooligosaccharides (FOS) for the production of organic acids was evaluated. Moreover, the sucA gene of R. microsporus was sequenced and its phylogenetic position was decided.

2. Materials and Methods

2.1. Strains and Culture Conditions The following Rhizopus strains were obtained from the NITE Biological Research Center (Chiba, Japan): Rhizopus microsporus var. chinensis NBRC 4737, 4768, 31988; R. microsporus var. microsporus NBRC 32995, 32996; R. microsporus var. rhizopodiformis NBRC 32997; R. microsporus var. tuberosus NBRC 100014; R. stolonifer var. lyococcus NBRC 32998; R. stolonifer var. stolonifer NBRC 4781, 5411, 6188, 30816. Cells were grown on potato dextrose agar (Becton Dickinson Company, Franklin Lakes, NJ, USA), which was prepared horizontally in a standing test tube. Mycelia were collected and used to inoculate 100 mL of liquid medium composed of 10% sucrose or short-chain FOS (mixture of 1-kestose, nystose, and 1-fructofuranosyl-D-nystose) [13] as the carbon source, as well as 0.2% (NH4)2SO4, 0.065% KH2PO4, 0.025% MgSO4, and 2.5% CaCO3 in a 300 mL Erlenmeyer ﬂask. Cultures were incubated at 30 ◦C with shaking (150 rpm).

2.2. Analyses of Organic Acids Production and Sugar Hydrolysis A portion of each culture was withdrawn and, after centrifugation at 3000× g for 15 min at 4 ◦C, the amounts of organic acid in the supernatant was determined by a high-performance liquid chromatography system (LaChrom Elite, Hitachi High Technologies, Tokyo, Japan) equipped with a packed column (Shodex RSpak KC-811; Showa Denko Co., Tokyo, Japan) and ultraviolet (UV) monitor (SPD-10AVP, SIMADZU, Kyoto, Japan). The hydrolytic activities against sugars were analyzed by thin-layer chromatography (TLC) using a solvent system of 1-butanol:2-propanol:acetic acid:water (7:5:2:4, v/v). Each spot of sugar was visualized by spraying with anisaldehyde-sulfuric acid reagent according to the method of Watanabe and Oda [7].

2.3. Preparation of Cell-Free Extracts Fungal cells were harvested by centrifugation at 3000× g for 5 min, and cell pellets were stored at −80 ◦C until use. Harvested cell pellets were washed with 0.1 M potassium phosphate buffer (pH 7.4) to remove residual medium components and resuspended in the same fresh buffer. The cell suspension was disrupted by sonication (pulse on, 3 s; pulse off, 5 s; power, 40%) on ice for 10 min using a model JY 92-IIN sonicator (Scientz, Ningbo, China) and centrifuged at 6000× g for 15 min at 4 ◦C. The supernatant was used as a cell-free extract for the following enzyme assays.

2.4. Enzyme Assays Sucrose-hydrolyzing activity was determined according to the method of Oda and Tonomura [14]. Briefly, a portion of the cell-free extract was added to 100 mM acetate buffer (pH 5.0) containing 150 mM sucrose to a final volume of 0.25 mL. After incubation at 30 ◦C for 30 min, the reaction was stopped by the addition of 1.0% (w/v) 3,5-dinitrosalicylic acid reagent, and the amount of reducing sugar produced was determined by the method of Oda and Tonomura [14]. One unit was defined as the amount of enzyme that released 1 µmol of reducing sugar equivalent to glucose per minute under the above conditions.

2.5. Identiﬁcation and Sequencing of sucA Gene Chromosomal DNA was extracted from lyophilized cells of each strain according to the methods of Sone et al. [15] and puriﬁed using the ZR Fungal/Bacterial DNA kit (ZYMO Research, Irvine, CA, USA). Microorganisms 2018, 6, 26 3 of 11

Oligonucleotide primers (RD_dPCR_INV_F1, 50-TGGATGAAYGAYCCIAAYGGIYTITTYTAYGA-30; RD_dPCR_INV_R1, 50-TAYTGCCARTTISWIGCCCAIGCIARNCC-30) were designed by Primer3Plus using the conserved sequences of domains A and E in the SucA of R. delemar NBRC 4754 (AB701479), Amylomyces rouxii CBS 436.76 (AB701482), and Schwanniomyces occidentalis ATCC 26077 (ADN34605), respectively. Degenerate PCR was performed for the detection of the sucA gene under the following ampliﬁcation conditions using a thermal cycler (T100™ Thermal Cycler; Bio-Rad, Hercules, CA, USA): denaturation at 96 ◦C for 1 min, annealing at 50 ◦C for 30 s, and extension at 72 ◦C for 2 min. The whole genome sequencing was performed with a next-generation sequencer (MiSeq system; Illumina, San Diego, CA, USA) using the paired-end mode (300 bp × 2) in combination with the software program Tablet for next-generation sequence assembly [16]. The DNA sequence of the sucA gene was identiﬁed using the BioEdit multiple sequence alignment editor (Ibis Biosciences, Carlsbad, CA, USA) and the BLAST database.

3. Results

3.1. Detection of β-Fructofuranosidase (sucA) Gene and Production of Organic Acids and Ethanol in Rhizopus Strains Degenerate oligonucleotide primers were designed based on a conserved region closely related to the sequence of the fungal sucA gene, and PCR was performed using these degenerate primers. The presence/absence of sucA genes among the seven R. microsporus strains and five R. stolonifera strains tested is shown in Table1. DNA amplification occurred only in R. microsporus NBRC 32995, indicating that among the Rhizopus strains tested, only this strain carries the sucA gene. The amplified DNA fragment was approximately 1.6 kbp according to gel electrophoresis. The Rhizopus strains were incubated in liquid medium containing sucrose as a carbon source at 30 ◦C for 14 days, and the production levels of organic acids (malic acid, lactic acid, and fumaric acid) and ethanol were evaluated (Table1). R. microsporus NBRC 32995 produced the highest levels of all organic acids and ethanol among all R. microsporus strains tested. In particular, the production of lactic acid was significantly higher than that of the other organic acids, reaching approximately 58.1 mg/mL of culture. Among R. stolonifer strains, the production of all organic acids and ethanol was the highest for R. stolonifer NBRC 4781, and the production level of lactic acid was significantly higher than those of the other organic acids. While the production level of lactic acid from R. microsporus NBRC 32995 was lower than those from R. stolonifer NBRC 4781 and R. oryzae NBRC 4785, only R. microsporus NBRC 32995 carries the sucA gene. Microorganisms 2018, 6, 26 4 of 11

Table 1. Detection of sucA gene and the production of organic acids and ethanol in Rhizopus strains.

Concentration (mg/mL) Strains sucA 1 References Malic Acid Lactic Acid Fumaric Acid Ethanol Rhizopus oryzae NBRC 4785 − 6.34 ± 1.53 93.67 ± 14.46 0.72 ± 0.45 5.80 ± 1.90 Watanabe and Oda [7] Rhizopus delemar NBRC 4754 + 34.26 ± 10.03 8.18 ± 1.81 30.28 ± 9.74 30.71 ± 9.36 Orikasa and Oda [8] R. microsporus var. chinensis NBRC 4737 − n.d. 2 0.25 ± 0.10 n.d. 0.24 ± 0.10 This study R. microsporus var. chinensis NBRC 4768 − n.d. 0.11 ± 0.07 n.d. 1.44 ± 0.56 This study R. microsporus var. chinensis NBRC 31988 − n.d. 1.00 ± 0.71 n.d. 1.91 ± 0.58 This study R. microsporus var. microsporus NBRC 32995 + 32.71 ± 6.42 58.06 ± 6.36 7.87 ± 1.41 19.60 ± 0.66 This study R. microsporus var. microsporus NBRC 32996 − 0.44 ± 0.11 0.31 ± 0.05 n.d. 0.41 ± 0.07 This study R. microsporus var. rhizopodiformis NBRC 32997 − n.d. n.d. n.d. 0.06 ± 0.01 This study R. microsporus var. tuberosus NBRC 100014 − n.d. 0.18 ± 0.07 n.d. 0.15 ± 0.03 This study R. stolonifer var. lyococcus NBRC 32998 − 2.44 ± 0.58 n.d. n.d. 0.14 ± 0.04 This study R. stolonifer var. stolonifer NBRC 4781 − 9.24 ± 1.50 81.26 ± 10.41 5.63 ± 1.32 14.32 ± 1.74 This study R. stolonifer var. stolonifer NBRC 5411 − 0.93 ± 0.41 0.25 ± 0.06 n.d. 0.23 ± 0.04 This study R. stolonifer var. stolonifer NBRC 6188 − 1.32 ± 0.41 0.47 ± 0.22 n.d. 2.29 ± 0.39 This study R. stolonifer var. stolonifer NBRC 30816 − 1.78 ± 0.34 0.26 ± 0.08 n.d. 1.36 ± 0.32 This study 1 PCR ampliﬁcation of sucA gene; +, detected; −, not detected. 2 not detected. Microorganisms 2018, 6, x FOR PEER REVIEW 5 of 10

Microorganisms 2018, 6, 26 5 of 11 3.2. Analysis of Sucrose and FOS Hydrolysis by TLC 3.2. AnalysisThe hydrolysis of Sucrose of and sucrose FOS Hydrolysisand FOS by by R. TLC microsporus NBRC 32995 was compared with those of R. delemarThe hydrolysis NBRC 4754 of sucroseand R. oryzae and FOS NBRC by R.4785 microsporus (Figure 1).NBRC For all 32995 strains was tested, compared according with thoseto TLC, of R.the delemar spot ofNBRC sucrose 4754 almost and R.disappeared, oryzae NBRC and 4785 new (Figure spots 1corresponding). For all strains to tested,monosaccharide according emerged to TLC, theafter spot 10 days of sucrose of incubation, almost disappeared, indicating that and these new strains spots corresponding have the ability to to monosaccharide decompose sucrose emerged into aftermonosaccharides 10 days of incubation, (Figure 1A,C,E). indicating The that spots these corre strainssponding have to the monosaccharides ability to decompose produced sucrose byinto the monosaccharidesdecomposition of (Figuresucrose1 A,C,E).mostly Thedisappeared spots corresponding after 21 days to of monosaccharides incubation for both produced R. microsporus by the decompositionNBRC 32995 and of sucrose R. delemar mostly NBRC disappeared 4754, but after some 21 daysmonosaccharides of incubation forremained both R. microsporusup to 28 daysNBRC of 32995incubation and R. for delemar R. oryzaeNBRC NBRC 4754, 4785 but (Figure some monosaccharides 1E). Moreover, for remained both R. up microsporus to 28 days NBRC of incubation 32995 and for R.R. oryzaedelemarNBRC NBRC 4785 4754, (Figure almost1E). all Moreover, FOS spots for were both significantlyR. microsporus reducedNBRC in 32995 intensity and afterR. delemar 10 daysNBRC and 4754,had completely almost all FOSdisappeared spots were after signiﬁcantly 28 days of incubation reduced in (Figure intensity 1B,D). after However, 10 days and for hadR. oryzae completely NBRC disappeared4785, FOS spots after remained 28 days of and incubation their intensities (Figure1 B,D).were However,maintained for toR. some oryzae extentNBRC up 4785, to 28 FOS days spots of remainedincubation and (Figure their 1F). intensities These results were maintained demonstrate to that some R. extent microsporus up to 28NBRC days 32995 of incubation as well as (Figure R. delemar1F). TheseNBRC results 4754 have demonstrate the ability that to hydrolyzeR. microsporus bothNBRC sucrose 32995 and FOS as well into as monosaccharides,R. delemar NBRC 4754indicating have thethe abilitycomplete tohydrolyze hydrolysis both of FOS sucrose by β and-fructofuranosid FOS into monosaccharides,ase; however, indicating R. oryzae theNBRC complete 4785 did hydrolysis not show of FOSthe ability by β-fructofuranosidase; to hydrolyze FOS. however, R. oryzae NBRC 4785 did not show the ability to hydrolyze FOS.

FigureFigure 1.1. Thin-layerThin-layer chromatography (TLC) analyses of residualresidual sugarssugars inin mediamedia containingcontaining sucrosesucrose ((AA,,BB,,EE)) oror fructooligosaccharidesfructooligosaccharides ((BB,,DD,,FF)) asas thethe carboncarbon sourcesource duringduring thethe incubationincubation ofof R. microsporus NBRCNBRC 32995 ( (AA,,BB),), R.R. delemar delemar NBRCNBRC 4754 4754 (C (,CD,),D and), and R. R.oryzae oryzae NBRCNBRC 4785 4785 (E,F). (E A,F ).portion A portion of each of eachculture culture was withdrawn, was withdrawn, and the and residual the residual sugars sugars in the insupernatant the supernatant were separated were separated by TLC. by Spots TLC. Spotscorresponding corresponding to sucrose to sucrose and fructooligosaccharides and fructooligosaccharides are indicated are indicated by single by (*) single and double (*) and asterisks double asterisks (**), respectively. GF2, 1-kestose; GF3, nystose; GF4, 1-fructofuranosyl-D-nystose. (**), respectively. GF2, 1-kestose; GF3, nystose; GF4, 1-fructofuranosyl-D-nystose.

3.3.3.3. Sucrose-Hydrolyzing ActivityActivity andand pHpH inin thethe CultureCulture MediumMedium FigureFigure2 2 shows shows the the sucrose-hydrolyzing sucrose-hydrolyzing activities activities and and culture culture pH pH values values during during the the incubation incubation ofof R. microsporus microsporus NBRCNBRC 32995, 32995, R.R. delemar delemar NBRCNBRC 4754, 4754, and and R. oryzaeR. oryzae NBRCNBRC 4785. 4785. The pH The values pH values in all incultures all cultures decreased decreased during during incubation incubation owing to owing the re tolease the of release organic of acids organic into acids the medium into the (Table medium 1).

Microorganisms 2018, 6, 26 6 of 11

(Table1). Under the condition of low pH, the sucrose-hydrolyzing activity of R. microsporus NBRC Microorganisms 2018, 6, x FOR PEER REVIEW 6 of 10 32995 appeared to increase during incubation, with the level at 14 days being approximately twice that at 10Under days the (approximately condition of low 0.1 pH, U/mg the sucrose-hydrolyzing protein) (Figure 2activityA). R. of delemar R. microsporusNBRC 4754NBRCalso 32995 exhibited sucrose-hydrolyzingappeared to increase activity during that incubation, increased with during the level incubation at 14 days tobeing approximately approximately 0.37twice U/mgthat at protein at 14 days10 ofdays incubation (approximately (Figure 0.1 U/mg2B), indicatingprotein) (Figure that 2A).R. R. microsporus delemar NBRCNBRC 4754 also 32995 exhibited as well sucrose- as R. delemar hydrolyzing activity that increased during incubation to approximately 0.37 U/mg protein at 14 days NBRC 4754of incubation have the (Figure ability 2B), to hydrolyzeindicating that sucrose R. microsporus even at NBRC a low 32995 pH. as However, well as R. delemarR. oryzae NBRCNBRC 4754 4785 did not showhave sucrose-hydrolyzing the ability to hydrolyze activity sucrose ateven either at a low 10 orpH. 14 However, days of R. incubation oryzae NBRC under 4785 did a lownot show pH. The pH value insucrose-hydrolyzing the R. oryzae NBRC activity 4785 at cultureeither 10 wasor 14 reduceddays of incubation to approximately under a low pH. 3.6 The by pH day value 10 ofin the incubation (Figure2C).R. oryzae Since NBRC the ability4785 culture of glucoamylase was reduced to toapproximately hydrolyze 3.6 sucrose by day drops10 of incubation by approximately (Figure 2C). 90% at Since the ability of glucoamylase to hydrolyze sucrose drops by approximately 90% at pH 3.5 [7], pH 3.5 [7], these results indicate that R. oryzae NBRC 4785 does not have β-fructofuranosidase activity these results indicate that R. oryzae NBRC 4785 does not have β-fructofuranosidase activity capable capable ofof hydrolyzinghydrolyzing sucrose sucrose under under a low a low pH. pH. These These results results support support the finding the ﬁnding that the thatsucA the genesucA is gene is present inpresent both inR. both microsporus R. microsporusNBRC NBRC 32995 32995 and andR. R. delemar NBRCNBRC 4754 4754 but not but in not R. oryzae in R. NBRC oryzae 4785.NBRC 4785.

Figure 2.FigureSucrose-hydrolyzing 2. Sucrose-hydrolyzing activities activities and pHand values pH va oflues media of media during during the incubationthe incubation of R.of microsporusR. NBRC 32995microsporus (A); NBRCR. delemar 32995 (NBRCA); R. delemar 4754 NBRC (B);and 4754 (R.B); oryzaeand R. oryzaeNBRC NBRC 4785 4785 (C (C).). Cell-free Cell-free extracts extracts were were prepared, and the sucrose-hydrolyzing activities (bars: U/mg protein) were measured. Values prepared, and the sucrose-hydrolyzing activities (bars: U/mg protein) were measured. Values are the are the means of three replicates. N.D.: not detected. means of three replicates. N.D.: not detected.

3.4. Sequencing and Phylogenetic Analysis of β-Fructofuranosidase (sucA) Gene The sucA gene, encoding β-fructofuranosidase, of R. microsporus NBRC 32995 was ampliﬁed by degenerate PCR, and its complete nucleotide sequence was determined. The results showed that the full-length open reading frame (ORF) was 1590 bp and contained no introns (Figure S1). Microorganisms 2018, 6, x FOR PEER REVIEW 7 of 10

3.4. Sequencing and Phylogenetic Analysis of β-Fructofuranosidase (sucA) Gene The sucA gene, encoding β-fructofuranosidase, of R. microsporus NBRC 32995 was amplified by Microorganisms 2018, 6, 26 7 of 11 degenerate PCR, and its complete nucleotide sequence was determined. The results showed that the full-length open reading frame (ORF) was 1590 bp and contained no introns (Figure S1). Next, the Next,deduced the amino deduced acid amino sequence acid sequencewas obtained was obtainedbased on based the DNA on the sequence DNA sequence and subjected and subjected to a BLAST to a BLASThomology homology search. The search. SucA The protein SucA of protein R. microsporus of R. microsporus NBRC 32995NBRC exhibited 32995 exhibited high homology high homology with that withof R. thatmicrosporus of R. microsporus ATCC 62417,ATCC with 62417, an identity with an of identity 96%, and of 96%,also showed and also homology showed homology with that of with R. thatdelemar of R. NBRC delemar 4754NBRC (80%), 4754 Amylomyces (80%), Amylomyces rouxii CBS rouxii 436.76CBS (77%), 436.76 Schwanniomyces (77%), Schwanniomyces occidentalis occidentalis ATCC ATCC26077 26077(62%), (62%),and Debaryomyces and Debaryomyces hansenii hansenii CBSCBS 767 767(61%). (61%). The The SucA SucA protein protein has has three three conserved domains, A, D, and E, belonging to the glycoside hydrolase family [[17].17]. The presence of all domains was conﬁrmedconfirmed ininR. R. microsporusmicrosporusNBRC NBRC 32995 32995 (Figure (Figure3). 3). In In addition, addition, the the amino amino acid acid sequence sequence of theof the A domainA domain in R. in microsporus R. microsporusNBRC NBRC 32995 was32995 closely was relatedcloselyto related that of R.to delemarthat of NBRCR. delemar 4754. NBRC Interestingly, 4754. theInterestingly, A domain oftheR. A microsporus domain NBRCof R. microsporus 32995 includes NBRC eight 32995 amino includes acid residues eight positioned amino acid upstream residues of thepositioned MNDPNG upstream sequence of thatthe MNDPNG have been deletedsequence from that the have A domain been deleted of R. microsporus from the ATCCA domain 62417 of [18 R.] (Figuremicrosporus3). ATCC 62417 [18] (Figure 3).

Figure 3. Conserved domains A, D, and E from microbial β-fructofuranosidases in clade VIa. Figure 3. Conserved domains A, D, and E from microbial β-fructofuranosidases in clade VIa. Numbers in parentheses indicate positions in the enzyme from R. oryzae. Dots indicate identical Numbers in parentheses indicate positions in the enzyme from R. oryzae. Dots indicate identical amino amino acid residues. acid residues.

A dendrogramdendrogram was was created created using using the neighbor-joiningthe neighbor-joining method method based onbased the amino on the acid amino sequences acid ofsequences microbial ofβ microbial-fructofuranosidases β-fructofuranosidases (Figure4). This(Figure showed 4). This that showed the SucA that proteins the SucA of R.proteins microsporus of R. NBRCmicrosporus 32995 NBRC and R.32995 microsporus and R. microsporusATCC 62417 ATCC belong 62417 to belong clade VIato clade and VIa are and closely are closely related related to the βto-fructofuranosidases the β-fructofuranosidases of R. delemar of R. delemarNBRC NBRC 4754 and 4754Amylomyces and Amylomyces rouxii CBSrouxii 436.76. CBS 436.76. In addition, In addition, while thesewhile threetheseRhizopus three Rhizopusstrains strains belong belong to the Zygomycota,to the Zygomycota, their SucA their proteinsSucA proteins are closely are closely related related to the βto-fructofuranosidases the β-fructofuranosidases of Schwanniomyces of Schwanniomyces occidentalis occidentalisATCC 26077 ATCC and 26077Debaryomyces and Debaryomyces hansenii CBS hansenii 767, yeastsCBS 767, that yeasts belong that to the belong Ascomycota to the (Saccharomycotina),Ascomycota (Saccharomycotina), and are distantly and related are distantly to Rhizopus relatedspecies to (FigureRhizopus4). species (Figure 4).

Microorganisms 2018, 6, 26 8 of 11 Microorganisms 2018, 6, x FOR PEER REVIEW 8 of 10

Figure 4.Figure Phylogenetic 4. Phylogenetic analyses analyses of ofthe the am aminoino acid sequencessequences of microbialof microbialβ-fructofuranosidases. β-fructofuranosidases. A A dendrogramdendrogram was constructed was constructed using using the the neighbor-joi neighbor-joiningning method method from from the aminothe am acidino sequences acid sequences of of microbial β-fructofuranosidases belonging to clade VIa. The length of each branch indicates amino microbial β-fructofuranosidases belonging to clade VIa. The length of each branch indicates amino acid sequence divergence. Bootstrap values (>800), calculated from 1000 replications, are shown. The acid sequencenumbers divergence. in parentheses Bootst indicaterap the values accession (>800), number. calculated from 1000 replications, are shown. The numbers in parentheses indicate the accession number. 4. Discussion 4. DiscussionThe results showed that all Rhizopus strains tested have the ability to produce organic acids (lactic Theacid, results malic showed acid, and/or that fumaric all Rhizopus acid) and/or strains ethanol tested in mediumhave the with ability sucrose to as produce a carbon source,organic acids indicating that these Rhizopus strains are capable of sucrose degradation for the production of organic (lactic acid,acids malic and ethanol. acid, and/or In particular, fumaric the levels acid) of and/or these compounds ethanol in produced medium by R.with microsporus sucroseNBRC as a carbon source, indicating32995 and R. that stolonifer theseNBRC Rhizopus 4781 were strains significantly are capable higherthan of sucrose those produced degradation by the other for Rhizopusthe production of organicstrains acids tested and and ethanol. comparable In particular, to those produced the levels by R. of oryzae theseNBRC compounds 4785 and R.produced delemar NBRC by R. 4754, microsporus NBRC 32995which and were R. tested stolonifer previously NBRC [7,8 4781]. However, were PCRsignificantly using degenerate higher primers, than those which produced were designed by the other Rhizopusby strains the conserved testedsequences and comparable of domains to A andthose E in produced SucA, indicated by R. that oryzae DNA amplificationNBRC 4785 occurred and R. delemar only in R. microsporus NBRC 32995 among the Rhizopus strains tested (Table1). Additionally, PCR NBRC 4754,amplification which waswere also tested conducted previously using another [7,8]. forward However, primer PCR designed using from degenerate the conserved primers, region which were designedof domain by D inthe a set conserved of the same reversesequences primer. of The domains results showed A and that E no in amplification SucA, indicated occurred forthat DNA amplificationthe sucA occurredminus strains, only whichin R. amountedmicrosporus to 11NBRC strains 32995 tested among in this study. the Rhizopus Actually, allstrainssucA minus tested (Table 1). Additionally,strains in TablePCR1 amplificationdid not show sucrose-hydrolyzing was also conduc activityted using based another on β-fructofuranosidase forward primer (data designed not from the conservedshown). region These results of domain indicated D thatin a thesetsucA of thegene same was presentreverse only primer. in R. microsporus The resultsNBRC showed 32995 that no among the 12 Rhizopus strains tested, and that the sucA gene is not common among Rhizopus strains amplification(Table1 occurred). for the sucA minus strains, which amounted to 11 strains tested in this study. Actually, allThe sucA results minus of TLC strains analyses in showedTable 1 that didR. not microsporus show NBRCsucrose-hydrolyzing 32995 as well as R. delemaractivityNBRC based on β- fructofuranosidase4754 had the ability(data tonot hydrolyze shown) both. These sucrose results and FOSindicated into monosaccharides, that the sucA and gene that was this present process only in R. microsporuwas almosts NBRC complete 32995 by 28among days of the incubation. 12 Rhizopus In contrast, strainsR. oryzae tested,NBRC and 4785 that could the not sucA hydrolyze gene is not commonFOS among (Figure Rhizopus1). These strains results (Table support 1). the findings that both R. microsporus NBRC 32995 and R. delemar NBRC 4754 carry the sucA gene, encoding β-fructofuranosidase, while R. oryzae NBRC 4785 Thedoes results not (Tableof TLC1). analyses Since both showedR. microsporus that R.NBRC microsporus 32995 and NBRCR. delemar 32995NBRC as well 4754 as produce R. delemar the NBRC 4754 hadSucA the proteinability (βto-fructofuranosidase), hydrolyze both sucrose which degrades and FOS FOS into from monosaccharides, the terminal fructose and residue, that these this process was almostresults complete demonstrate by 28 that days the of complete incubation. hydrolysis In contrast, of FOS in R. these oryzae fungal NBRC strains 4785 is induced could not by the hydrolyze FOS (Figureβ-fructofuranosidase 1). These results secreted support from the their findings cells. However, that bothR. oryzae R. microsporusNBRC 4785 NBRC does not 32995 produce and this R. delemar NBRC 4754hydrolytic carry enzyme.the sucA Instead, gene,R. encoding oryzae is known β-fructofuranosidase, to degrade sucrose with while a glucoamylase, R. oryzae NBRC and R. 4785 oryzae does not NBRC 4785 was shown to produce glucoamylase in a previous study [7]. The activity of glucoamylase, (Table 1). Since both R. microsporus NBRC 32995 and R. delemar NBRC 4754 produce the SucA protein (β-fructofuranosidase), which degrades FOS from the terminal fructose residue, these results demonstrate that the complete hydrolysis of FOS in these fungal strains is induced by the β- fructofuranosidase secreted from their cells. However, R. oryzae NBRC 4785 does not produce this hydrolytic enzyme. Instead, R. oryzae is known to degrade sucrose with a glucoamylase, and R. oryzae NBRC 4785 was shown to produce glucoamylase in a previous study [7]. The activity of glucoamylase, which degrades FOS from the terminal glucose residue, is inhibited at pH values below pH 4.0 [7]. The pH value in the culture of R. oryzae NBRC 4785 was reduced to approximately 3.6 by the release of organic acids following 10 days of incubation (Figure 2C). The rate of sucrose and FOS degradation by R. microsporus NBRC 32995 tended to be lower than that by R. delemar NBRC 4754 (Figure 1A–D): while both strains degraded most of the sucrose and

Microorganisms 2018, 6, 26 9 of 11 which degrades FOS from the terminal glucose residue, is inhibited at pH values below pH 4.0 [7]. The pH value in the culture of R. oryzae NBRC 4785 was reduced to approximately 3.6 by the release of organic acids following 10 days of incubation (Figure2C). The rate of sucrose and FOS degradation by R. microsporus NBRC 32995 tended to be lower than that by R. delemar NBRC 4754 (Figure1A–D): while both strains degraded most of the sucrose and FOS in the medium by 10 days of incubation, traces of sucrose at day 10 and FOS at day 21 remained for R. microsporus NBRC 32995 (Figure1A,B). It appears that this result was caused by differences in the sucrose-hydrolyzing activities of the strains. The enzyme activity of R. microsporus NBRC 32995 increased during the incubation period and reached approximately 0.1 U/mg protein at day 14 of incubation, but this was approximately 1/4 that of the R. delemar NBRC 4754 enzyme (Figure2A,B). Recently, it has been reported that some R. microsporus cells produce thermostable inulinase (EC 3.2.1.7) as a glycolytic enzyme [19], which is secreted as an extracellular enzyme [20]. However, based on the genome database, R. microsporus NBRC 32995 does not share genomic sequence homology with genes that encode the inulinase protein, indicating that the hydrolysis of FOS is not caused by inulinase. According to the dendrogram based on amino acid sequences, enzymes with β-fructofuranosidase activity can be classified into seven clades: the monocotyledonous clades I, II, IV, and V; the dicotyledonous clade III; the fungal clades VIa and VIb; and the bacterial clade VII [9]. In a previous study, the SucA proteins from R. delemar and A. rouxii were classified into clade VIa [8]. This clade has a conserved sequence of H-[FY]-[ST]-P-x-[KS]-[NG]-[WF]-MNDPNG in domain A and a signal peptide upstream of this region (Figure3), suggesting the possibility of enzyme secretion outside the cell [9]. The SucA protein of R. microsporus NBRC 32995 also contained domain A belonging to clade VIa, and its amino acid sequence was closely related to that of R. delemar NBRC 4754 (Figure3). In addition, the other two highly conserved regions of SucA, domains D and E, were also found in the SucA protein of R. microsporus NBRC 32995 and also belonged to this clade. The SucA full-length amino acid sequence of R. microsporus NBRC 32995 (530 amino acids) exhibited high homology (approximately 80% identity) with that of R. delemar NBRC 4754. These results indicate that the newly identified SucA protein of R. microsporus NBRC 32995 should be classified as a clade VIa member (Figures3 and4). The amino acid sequences of SucA in the conserved regions of domains D and E were identical between R. microsporus NBRC 32995 and R. microsporus ATCC 62417. However, an eight-amino acid sequence of HYTPEKGW, positioned upstream of the MNDPNG sequence in domain A of R. microsporus NBRC 32995, was deleted in the R. microsporus ATCC 62417 sequence (Figure3), implying that R. microsporus ATCC 62417 may not possess proper β-fructofuranosidase activity. Together, these results suggest that R. microsporus NBRC 32995, carrying a full-length sucA gene, is a unique strain even among R. microsporus and that its sucA gene may have been introduced into this strain by horizontal gene transfer. In a previous study, the possibility of the horizontal gene transfer of the sucA gene into Rhizopus was discussed [8]. Gene transfer is likely to occur through contact with other microorganisms such as yeast during the production of fermented foods [7]. However, it remains unclear whether this is the mechanism by which R. microsporus NBRC 32995 acquired its sucA gene, as this strain was isolated from chaff and its habitat appears to be distinct from those of other sucA-carrying strains, such as R. delemar NBRC 4754. Recently, genomic analyses have been conducted for a wide variety of filamentous fungi including Rhizopus; thus, the distribution of the sucA gene may be further elucidated in the future. The results obtained in this study show that R. microsporus NBRC 32995 is a unique Rhizopus strain carrying the full-length amino acid sequence of a SucA protein belonging to clade VIa that is closely related to the SucA proteins of yeasts (Ascomycota), suggesting that other Rhizopus species do not have SucA proteins closely related to those of Ascomycota. In addition, R. microsporus NBRC 32995 possesses β-fructofuranosidase activity and is capable of hydrolyzing sucrose, along with the production of organic acids (i.e., lactic acid, fumaric acid, and malic acid), even under low pH conditions. Such organic acids are used as raw materials for industrial purposes, such as flavor control Microorganisms 2018, 6, 26 10 of 11 in food processing. Thus, R. microsporus NBRC 32995 is of great potential value for the simultaneous production of multiple useful organic acids and thus may be useful for industrial applications.

Supplementary Materials: The following are available online at www.mdpi.com/2076-2607/6/1/26/s1, Figure S1: The full-length open reading frame (ORF) sequence of the sucA gene in R. microsporus NBRC 32995 (accession number: LC372541). Acknowledgments: We appreciate Kaho Kuroda and Yuko Nasu for their aid in analyzing the components of organic acids and measuring the hydrolytic enzyme activities. We also would like to thank Tsuyoshi Watanabe, lecturer at Kushiro Junior College, for his advice on the culture and classification of fungi. Author Contributions: Y.Or. and Y.Od. designed the experiments; Y.Or. and T.O. performed the experiments and wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Severo, C.B.; Guazzelli, L.S.; Severo, L.C. Chapter 7: Zygomycosis. J. Bras. Pneumol. 2010, 36, 134–141. [CrossRef][PubMed] 2. Hesseltine, C.W. Microbiology of oriental fermented foods. Ann. Rev. Microbiol. 1983, 37, 575–601. [CrossRef] [PubMed] 3. Rehms, H.; Barz, W. Degradation of stachyose, raffinose, melibiose and sucrose by different tempe-producing Rhizopus fungi. Appl. Microbiol. Biotechnol. 1995, 44, 47–52. [CrossRef][PubMed] 4. Abe, A.; Sone, T.; Sujaya, I.-N.; Saito, K.; Oda, Y.; Asano, K.; Tomita, T. rDNA ITS sequence of Rhizopus oryzae: Its application to classification and identification of lactic acid producers. Biosci. Biotechnol. Biochem. 2003, 67, 1725–1731. [CrossRef][PubMed] 5. Abe, A.; Oda, Y.; Asano, K.; Sone, T. Rhizopus delemar is the proper name for Rhizopus oryzae fumaric-malic acid producers. Mycologia 2007, 99, 714–722. [CrossRef][PubMed] 6. NU-IUBMB.; Webb, E.C. Enzyme Nomenclature; Academic Press: San Diego, CA, USA, 1992; pp. 346–369; ISBN 0-12-227164-5. 7. Watanabe, T.; Oda, Y. Comparison of sucrose-hydrolyzing enzymes produced by Rhizopus oryzae and Amylomyces rouxii. Biosci. Biotechnol. Biochem. 2008, 72, 3167–3173. [CrossRef][PubMed] 8. Orikasa, Y.; Oda, Y. Molecular characterization of β-fructofuranosidases from Rhizopus delemar and Amylomyces rouxii. Folia Microbiol. 2013, 58, 301–309. [CrossRef][PubMed] 9. Alméciga-Díaz, C.J.; Gutierrez, A.M.; Bahamon, I.; Rodríguez, A.; Rodríguez, M.A.; Sánchez, O.F. Computational analysis of the fructosyltransferase enzymes in plants, fungi and bacteria. Gene 2011, 484, 26–34. [CrossRef][PubMed] 10. Schipper, M.A.A. A revision of the genus Rhizopus: I. The Rhizopus stolonifer-group and Rhizopus oryzae. Stud. Mycol. 1983, 25, 1–19. 11. Schipper, M.A.A.; Stalpers, J.A. A revision of the genus Rhizopus: II. The Rhizopus microsporus group. Stud. Mycol. 1984, 25, 20–34. 12. Kitpreechavanich, V.; Maneeboon, T.; Kayano, Y.; Sakai, K. Comparative characterization of L-lactic acid-producing thermotolerant Rhizopus fungi. J. Biosci. Bioeng. 2008, 106, 541–546. [CrossRef][PubMed] 13. Oda, Y.; Ouchi, K. Construction of a sucrose-fermenting baker’s yeast incapable of hydrolyzing fructooligosaccharides. Enzyme Microb. Technol. 1991, 13, 495–498. [CrossRef] 14. Oda, Y.; Tonomura, K. Purification and characterization of invertase from Torulaspora pretoriensis YK-1. Biosci. Biotechnol. Biochem. 1994, 58, 1155–1157. [CrossRef] 15. Sone, T.; Abe, T.; Yoshida, N.; Suto, M.; Tomita, F. DNA fingerprinting and electrophoretic karyotyping of Japanese isolates of rice blast fungus. Ann. Phytopathol. Soc. Jpn. 1997, 63, 155–163. [CrossRef] 16. Milne, I.; Bayer, M.; Cardle, L.; Shaw, P.; Stephen, G.; Wright, F.; Marshall, D. Tablet-next generation sequence assembly visualization. Bioinformatics 2009, 26, 401–402. [CrossRef][PubMed] 17. Cantarel, B.L.; Coutinho, P.M.; Rancurel, C.; Bernard, T.; Lombard, V.; Henrissat, B. The carbohydrate-active enzymes database (CAZy): An expert for glycogenomics. Nucl. Acids Res. 2009, 37, 233–238. [CrossRef] [PubMed] Microorganisms 2018, 6, 26 11 of 11

18. Horn, F.; Guthke, R.; Hertweck, C. Draft genome sequences of symbiotic and nonsymbiotic Rhizopus microsporus strains CBS 344.29 and ATCC 62417. Genome Announc. 2015, 3, 14–15. [CrossRef][PubMed] 19. Flores-Gallegos, A.C.; Contreras-Esquivel, J.C.; Morlett-Chávez, J.A.; Aguilar, C.N.; Rodríguez-Herrera, R. Comparative study of fungal strains for thermostable inulinase production. J. Biosci. Bioeng. 2015, 119, 421–426. [CrossRef][PubMed] 20. Ohta, K.; Suetsugu, N.; Nakamura, T. Puriﬁcation and properties of an extracellular inulinase from Rhizopus sp. strain TN-96. J. Biosci. Bioeng. 2002, 94, 78–80. [CrossRef]

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