Annals of Microbiology (2018) 68:881–888 https://doi.org/10.1007/s13213-018-1394-3

ORIGINAL ARTICLE

High alkaline activity of a thermostable α- (cyclomaltodextrinase) from thermoacidophilic Alicyclobacillus isolate

Lina Zhang1 & Huijia Yin2 & Qi Zhao2 & Chunyu Yang2 & Yan Wang1

Received: 4 July 2018 /Accepted: 24 October 2018 /Published online: 1 November 2018 # Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018

Abstract It is well demonstrated that glycosyl from Alicyclobacillus strains are general thermoacidophilic and are ideal proteins for industrial applications. In this study, a thermophilic Alicyclobacillus α-amylase of glycoside hydrolases 13_20 subfamily, AMY1, was identified from an Alicyclobacillus strain and efficiently expressed in the host Escherichia coli BL21 CodonPlus. In agreement with other reported Alicyclobacillus hydrolases, the purified AmyY1 had an optimal pH of 6.0–6.5 in phosphate or citrate/Na2HPO4 buffers, and a remarkably decreased activity at pH 8.0. Differently, much higher activity was detected in the alkaline glycine/NaOH reaction mixtures. Compared to the highest amylolytic activity at pH 6.0, AmyY1 exhibited 230 and 116% activities at pH 8.0 and 9.0, respectively. This glycine-activation was further confirmed by a supple- mentation of glycine into the assay mixtures. During the digestions of various raw starches, AmyY1 also exhibited high hydrolysis efficiency under acidic or alkaline conditions. Findings in this study not only endow AMY1 with much broad applications, but also may provide a novel field for the application potentials of some other Alicyclobacillus hydrolases.

Keywords Alicyclobacillus α-amylase AMY1 . Alkaline activity . Glycine/NaOH solution . Raw starch hydrolysis

Introduction (CDs), pullulan, and starch (Stam et al. 2006; Kuchtová and Janeček 2016). Family 13 of the glycoside hydrolases (GH) is a present- Among these hydrolases, α- constitute a class ly huge group within the Carbohydrate-Active Enzymes of important industrial enzymes that are widely used in database (CAZy, www.cazy.org) database (Martinovičová various industries like food, detergent, textiles, and phar- and Janeček 2018). It comprises of more than 40 maceuticals (Sivaramakrishnan et al. 2006;Souzaand subfamilies and diverse hydrolyses including α- Magalhães 2010). During conventional starch processing, amylases (EC 3.2.1.1), neopullulanases (EC 3.2.1.135), starch slurry is first gelatinized by heating and then sub- cyclomaltodextrinases (EC 3.2.1.54), etc. With more than jected to two enzymatic steps—liquefaction (by α- one , the subfamily GH13_20 usually possess amylase) and saccharification (by glucamylase) (Mjec the N-terminal starch-binding domain (SBD) classified et al. 2002). Due to different pH requirements of these as the carbohydrate-binding module family CBM34 steps, complex pH adjustments are required. Therefore, (Machovic and Janeček 2006), and thus can hydrolyze amylases with high activity and stability at low pH values at least two of the three substrates cyclomaltodextrins are desirable for such industrial processes. In contrast, other applications such as textiles, detergents, and dishwashing machines require amylases that are active * Chunyu Yang and stable under alkaline pH values (Saxena et al. [email protected] 2007). Besides the varying pH requirements for amylolyt- * Yan Wang ic activities, thermostable α-amylases are another type of [email protected] attractive hydrolases for industry and research. As the

1 whole process for conventional enzymatic saccharification State Key Laboratory of Biobased Material and Green Papermaking, Qilu University of Technology, Shandong Academy of Sciences, requires high temperature (Prakash and Jaiswal 2010), Jinan 250353, People’s Republic of China thermostable amylases provide the advantages of decreas- 2 State Key Laboratory of Microbial Technology, Shandong ing the risk of contamination and cost of external cooling University, Qingdao 266237, People’sRepublicofChina as well as increasing the diffusion rate. 882 Ann Microbiol (2018) 68:881–888

Enzymatic degradation of raw starch granules is important adjusted to 4.0 with HCl. After incubated at 60 °C for 14 days, for industrial applications and consequently raw-starch- the culture was transferred into the same medium for next digesting amylases are ideal enzymes for these processes. enrichment. Culture from the third enrichment was diluted Therefore, there are considerable interests in the isolation of and spread onto M-5 agar plates. new strains, especially extremophiles, producing suitable am- ylases for raw-starch digestion (Dumorné 2018). Among Gene cloning and sequence analysis them, Aspergillus sp., Rhizopus sp., Bacillus sp., and Geobacillus sp. are apparently the most suitable producers Genomic DNA was isolated from strain Alicyclobacillus sp. with high hydrolysis efficiency (Sun et al. 2010; Mehta and HJ and used as template for 16S rDNA amplification and Satyanarayana 2014). genomic sequencing. 16S rDNA was amplified by PCR using Apart of these strains, members of genus Alicyclobacillus universal primers 27F and 1492R for bacteria (Lane 1991). are a recently attractive producer for thermophilic amylases. PCR was purified with the Qiagen ІІ Extraction Kit, Being the gram-positive, thermoacidophilic, heterotrophic ligated into pEASY-blunt vector, and transformed into E. coli organisms that mostly inhabit acidic geothermal environ- DH5α competent cells. ments such as hot springs (Simbahan et al. 2004), these Genomic DNA of Alicyclobacillus sp. HJ was sequenced strains are known to be valuable sources for many thermo- by the Illumina Hiseq2000 platform and annotated by submit- stable and acidic hydrolyses such as xylanase, α-amylase, ting to the Rast server. The genome analysis revealed an and endoglucanase (Bai et al. 2010; Kumar et al. 2010; ORF0788 fragment encoding anα-amylase (AMY1). Boyce and Walsh 2015). To our knowledge, these Multiple alignments were conducted using the ClustalX pro- Alicyclobacillus hydrolases are general reported for their gram (Thompson et al. 1997) by using some homologue genes optimally acidic activities and no data was documented for retrieved from the National Center for Biotechnology high activities under alkaline conditions. Information (NCBI) database. Due to the special habitat of Alicyclobacillus strains, the For intracellular expression, amplification was con- growth of these strains is slowly and requires complex medi- ducted by using primers 0788-F (5′–CTAGCTA um. Therefore, heterologous expression of their hydrolases is GCATGGTTCTTGTGTTGCGC–3′) and 0788-R (5′– essential for large-scale applications. Previously, a CdaA pro- GCGTCGACCTGGTTGTGAAATCCGTC–3′)whichin- tein was isolated from the wild-type strain Alicyclobacillus corporate restriction sites NheIandSalI, respectively. acidocaldarius and its biochemical properties had been inves- The PCR product was digested and ligated into the ex- tigated (Matzke et al. 2000). However, the heterologous ex- pression vector pET-24a(+) and the recombinant plasmid pression protocol and its potentials for industrial applications was transformed into E. coli BL21 CodonPlus. remained to be addressed. In the present study, α-amylase AMY1 was cloned from an Alicyclobacillus strain and suc- Protein expression and purification cessfully expressed in a codon bias-adjusted Escherichia coli host of BL21 CodonPlus. Interestingly, AMY1 was much Luria-Bertani (LB) medium with the additions of 50 μgmL−1 active in the alkaline glycine-buffered mixtures and implied kanamycin and 40 μgmL−1 chloromycetin was used for cell good potentials as a thermostable and alkaline α-amylase. cultivation. When cells reached to an optical density of 0.6

Furthermore, abilities of this α-amylase in digesting various (OD600), isopropyl β-D-1-thiogalactopyranoside (IPTG) was raw starches were also investigated under both acidic (pH 6.0) added to a final concentration of 0.5 mM to induce the protein. and alkaline (pH 9.0) conditions. To avoid excessive formation of inclusion bodies, cells were incubated at 16, 22, 30, or 37 °C for various time and the expression levels were visualized by 11.25% (w/v) sodium Materials and methods dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE). In detail, cells were collected by centrifugation at Strain isolation and identification 4000×g for 10 min at 4 °C, washed twice with PBS

(60 mM, Na2HPO4,KH2PO4, pH 7.4), and resuspended in Isolation was carried out with water samples collected from HisTrap buffer A (60 mM PBS, 10% glycerol (v/v), and acidic Tengchong hot springs. Enrichment was conducted in 0.1 mM PMSF, a protease inhibitor tablet, and 10 mM 2-

M-5 medium which consists of 3.7 mM KH2PO4, 11.6 mM mercaptoethanol, pH 7.4). The bacterial suspension was then Na2HPO4·7H2O, 13.4 mM KCl, 3.8 mM (NH4)2SO4,9.3mM passed twice through a French press at 15,000 psi and 4 °C −1 NH4Cl, 0.2 mM MgCl2·6H2O, 0.3 mM CaCl2·H2O, 1 g L (Aminco). After centrifugation at 12,000×g for 15 min, the skim milk, 3 g L−1 yeast extract, 5 mL minor elements solu- debris was solubilized in a same volume of PBS as that of tion, and 0.5 mL vitamin solution (Engle et al. 1995). Due to the supernatant. Both supernatant and solubilized debris were the hot spring sample was pH 4.0, the pH of the medium was subjected to SDS-PAGE analysis. Ann Microbiol (2018) 68:881–888 883

After 12-h incubation at 16 °C, cells were harvested and starch granules from potato, corn, sweet potato, or wheat was disrupted as above described. The supernatant was collected incubated with 20 U of AMY1 at 65 °C. Samples were and applied to a Ni-NTA HisTrap affinity column (GE taken interval and centrifuged at 10,000×g for 10 min at Healthcare, Uppsala, Sweden) in an AKTA Prime System 4 °C, then the reducing sugars in the supernatant were (GE Healthcare). AMY1 was eluted by a stepwise gradient quantified by DNS method and used glucose as a stan- of imidazolein Tris/HCl buffers (50–500 mM) and the dard. The extent of hydrolysis of raw starch (Rh)was resulting protein fractions were analyzed by SDS-PAGE. defined by the following formula: Rh (%) = (A1/A0)× Another PAGE gel running in the same condition was also 100, where A1 was the amount of sugar in the supernatant analyzed by western blot using His-tag antibody (Abcam) as after the hydrolysis reaction and A0 was the amount of we previously described (Yin et al. 2018). Protein sample was raw starch before the reaction (Liu and Xu 2008). also loaded on a native polyacrylamide and SDS-free gel with 1gL−1 soluble starch addition. After 90 min separation at 4 °C Nucleotide sequence accession number and 120 V, the gel was incubated in 50 mM glycine/NaOH buffer for 30 min at 65 °C and stained by Lugol’s iodine The nucleotide sequence for the α-amylase gene (AMY1) solution (Atlas 1993). was deposited in the GenBank database under accession number of KX355641. Biochemical properties of AMY1 Data availability All data generated or analyzed during this For routine analysis, the activity of purified AMY1 was deter- study are included in this published article. mined by measuring the amount of reducing sugar released during the enzymatic hydrolysis of 5 g L−1 soluble starch in tested buffers at 65 °C for 15 min. The reducing sugar was Results and discussion measured with a modified dinitrosalicylic acid method (Miller 1959). One unit of α-amylase activity was defined as the Strain isolation and α-amylase identification amount of that released 1 μmol of glucose per minute at 65 °C (Hagihara et al. 2001). Alicyclobacillus sp. HJ strain was isolated from the Tengchong Effect of pH on the α-amylase activity was determined in hot spring. 16S rDNA sequence analysis revealed 100% similar- various buffers ranging from 3.0 to 10.0 (50 mM of citrate/ ity with gene of A. acidocaldarius DSM451 (Goto et al. 2002).

Na2HPO4 buffer, pH 3.0–8.0; 50 mM of NaH2PO4/Na2HPO4, After incubated at 60 °C and stained with Lugol’s iodine solu- pH 5.6–8.0; 50 mM of Tris-HCl, pH 8.0–10.0; 50 mM of tion, big transparent zones were observed around the colonies on barbital sodium/HCl, pH 7.0–8.6; C8H18N2O4S (HEPES), a plate containing soluble starch. Draft genome sequence from pH 6.8–8.2; 50 mM of glycine/NaOH, pH 8.0–10.0), by main- Illumina Hiseq2000 identified an open reading frame taining a constant temperature of 65 °C. In addition, 50 mM of (ORF0788) of 578 aa that encoding putative α-amylase. It was

Na2B4O7/HCl buffers with pH ranging from 8.0 to 9.0 was closely related with the CdaA protein of A. acidocaldarius used for the alkaline activity of AMY1. To investigate the (CAB40078) (Matzke et al. 2000) and with a cyclodextrinase effect of glycine concentration on the activity, the amylolytic that isolated from hot spring enrichments in the southern part of activities was determined in glycine/NaOH solutions at pH 8.0 Iceland (Labes et al. 2008). Based on the CAZY database or 9.0 but with a supplementation of various concentration (Cantareletal.2009), these homologues belong to GH13_20 glycine. The pH stability was measured by analyzing the re- subfamily that comprising of α-amylase (EC 3.2.1.1), sidual activity after pre-incubating of the enzyme at 30 °C in (EC 3.2.1.41), cyclomaltodextrin glucanotransferase buffers of citrate/Na2HPO4 (pH 6.0) and glycine/NaOH (EC 2.4.1.19), cyclomaltodextrinase (EC 3.2.1.54), etc. (pH 9.0) for 2 h, respectively. For the temperature optima Sequence alignment shown in Fig. 1 indicated that AMY1 pos- measurement, the enzymatic activity was determined after in- sesses all the typical domain signatures of GH13_20 subfamily cubating the enzyme with soluble starch at different tempera- group, including the fingerprint sequences of 286_MPKLN in tures ranging from 55 to 80 °C in citrate/Na2HPO4 buffer the fifth conserved sequence region (CSR-V) and 315_VANE in (pH 6.0) or glycine/NaOH buffer (pH 9.0). Thermal stability the CSR-II region (Oslancová and Janeček 2002). Based on the was tested in the standard assay conditions after pre- CAZY classification, it has a typical carbohydrate-binding mod- incubating AMY1 at 65 °C for various time intervals. ule CBM34 region in its N-terminal as those positions in many GH13_20 proteins (Kuchtová and Janeček 2016), as well as the Raw starch hydrolysis crucial residue of Asp65 for starch-binding (Abe et al. 2004). As describe in its homologue protein CdaA (Matzke et al.

A 10-mL citrate/Na2HPO4 (pH 6.0) or glycine/NaOH 2000), AMY1 possessed a mixed specificity toward various (pH 9.0) buffered mixture containing 50 mg of various raw substrate (Table 1). The maximum hydrolysis efficiency was 884 Ann Microbiol (2018) 68:881–888

Fig. 1 Multiple sequence alignment of AMY1 with other GH13_20 cyclomaltodextrinase Bacillus sp.; O06988, maltogenic amylase hydrolases. APZ86803, AMY1, Alicyclobacillus sp. HJ, this study; Bacillus subtilis; Q08751, neopullulanase Thermoactinomyces vulgaris. P38940, neopullulanase Geobacillus stearothermophilus; Q046J8, The conserved residue of Asp is highlighted in red square and conserved maltogenic α-amylase Lactobacillus gasseri; Q9WX32, sequence characteristics of GH13_20 were also labeled cyclomaltodextrinase Alicyclobacillus acidocaldarius; Q59226, observed in the soluble starch, α-cyclomaltodextrin, and β- Overexpression and purification of AMY1 cyclomaltodextrin. Obviously, it showed a clear preference to cyclomaltodextrins and starch over pullulan, with only 21.2% It is well accepted that the expression of heterologous proteins hydrolysis rate obtained toward pullulan. This substrate spec- in E. coli is strongly affected by codon bias (Rosano and trum implies that AMY1 should be assigned as a Ceccarelli 2009). To our knowledge, available information cyclomaltodextrinase, α-amylase, or neopullulanase which for these hydrolases only focus on their catalytic properties, belongs to GH13_20 subfamily. while no detailed expression protocol described. Based on the Ann Microbiol (2018) 68:881–888 885

Table 1 Substrate spectrum of AmyY1. Reactions were performed in −1 corresponding position as shown in lane 5. Furthermore, glycine/NaOH buffer (pH 9.0) with 5 g L substrate additions, at 65 °C the expressed protein was highly active, with an intense for 15 min and bright band observed in the native gel (lane 4). This Substrate Relative activity (%) indicates effective soluble expressions of AMY1 and huge potentials of this protein in large-scale production. Soluble starch 100.0 ± 1.9a α-Cyclomaltodextrin 99.3 ± 2.2 β-Cyclomaltodextrin 100.0 ± 2.6 Optimal temperature and pH of AMY1 Potato starch 61.3 ± 2.8 Wheat starch 68.7 ± 2.3 As expected for an enzyme isolated from a thermophile, the Corn starch 54.5 ± 1.7 amylase exhibited the highest activity at 65 °C (Fig. 3a). Sweet potato starch 46.1 ± 2.6 Additionally, it was highly stable under acidic condition at Pullulan 21.2 ± 1.4 high temperature, with more than 90% residual activity detect- ed after an incubation at 65 °C for 120 min and above 80% a Soluble starch activity was regarded as 100% activity retained after 150-min incubation. Compared with the stability at pH 6.0, AMY1 also stable underalkaline condition, rare codon analysis of these sequences (http://people.mbi. with 86.1% residual activity detected after 120-min incubation ucla.edu/sumchan/caltor.html), a high content of rare codons, at pH 9.0 (Fig. 3b). e.g., 12.8% rare codons in the encoding sequence of AMY1, To explore the catalytic abilities of this thermostable α- and high frequencies of Arg, Gly, and Pro were detected. amylase, different pH buffer systems were used for its pH Therefore, E. coli BL21 CodonPlus was used as a host for profile evaluation. As shown in Fig. 4a, AMY1 displayed AMY1 expression and relative high soluble expression was an unexpected and interesting broad pH range for amylo- obtained. Further inductions at different temperatures lytic activity. In 50 mM sodium-phosphate buffers ranging showed that lower temperature was beneficial to the protein from pH 5.6 to 8.0, AMY1 exhibited the optimal activity solubility, with 57.9 mg soluble protein obtained from 1-L at pH 6.5. Similarity, the highest activity was observed at cell cultures after 16 °C induction for 12 h (Fig. 2a). On the pH 6.0 in the 50 mM citrate/Na2HPO4 buffers (pH 3.0– contrast, only less than half target protein was detected under 8.0) while very low activity was observed at pH 8.0. These higher temperatures. results were consistent with the pH profile of CdaA, which After stepwise gradient elution by imidazole, around showed the highest neopullulanase activity at pH 6.0 in

30.1 mg protein (91.2% purity) was eluted from His-strap. sodium/Na2HPO4 buffer (Matzke et al. 2000), as well as The purified amylase AMY1 displayed a clear protein band with the pH profile of many acidic Alicyclobacillus hydro- of around 60 kDa in SDS-PAGE (lane 3 in Fig. 2b), which lases (Kumar et al. 2010). Due to the potential applications was close to its estimated molecular of 64 kDa. The ex- of alkaline α-amylase in textile and detergent, the amylo- pression of AMY1 was also confirmed by western blot lytic activity of AMY1 was also investigated in some al- analysis, in which an intense band was observed at the kaline buffer systems. Interestingly, relative higher

Fig. 2 SDS-PAGE and western blot analysis for the expression of α- spectra of AMY1. Lane M, molecular mass markers; lane 1, cell extract; amylase AMY1 in E. coli BL21 CodonPlus. a Expression spectra of lane 2, cell debris; lane 3, purified AMY1 visualized by Coomassie AMY1 at different temperatures. Lane M, molecular mass markers; brilliant blue staining; lane 4, AMY1 extract visualized by Lugol’s lane 1, 16 °C; lane 2, 22 °C; lane 3, 30 °C; lane 4, 37 °C; b purification iodine staining; lane 5, AMY1 extract analysis by western blot 886 Ann Microbiol (2018) 68:881–888

Fig. 3 Effect of temperature on AMY1 activity and stability. a Temperature profile of AMY1 at pH 6.0 and pH 9.0; b Activities of AMY1 after being pre-incubated at 65 °C for various times in the citrate/Na2HPO4 buffer (pH 6.0) or glycine/NaOH buffer (pH 9.0) solutions. The observed maximal activity was defined as 100%

activities were detected in the HEPES or glycine-buffered that glycine can bind to proteins and structurally stabilize mixtures. The maximum activity was detected in the mix- the enzyme. The glycine-assisted enhancement of AMY1 ture that buffered with HEPES of pH 7.0. On the contrast, agrees with a xylanase XylI from a Thermomonospora sp., under a higher alkalinity of pH 9.0, AMY1 exhibited the in which a novel possible mechanism for the glycine- highest activity in the glycine/NaOH buffer. At pH 8.0 and assisted catalytic action of xylanase is proposed 9.0, the enzyme exhibited 230 and 116% of the activity (Vathipadiekal et al. 2007). Based on our preliminary compared to that of the optimal acidic pH (pH 6.0), re- study, the glycine concentration in the reaction mixture spectively. On the contrast, all the other alkaline systems showed an obvious decrease after 15-min incubation with of HEPES, Tris/HCl, and barbital/HCl displayed much the enzyme, while remained stable in the absence of weak alkaline activities (Fig. 4a). Due to the observed high AMY1 (data not shown). Therefore, it was suspected that activity in the alkaline glycine/NaOH buffers, the effect of glycine is also structurally involved in the enzyme cataly- glycine concentrations on the amylase activity of AMY1 sis at alkaline pH, which accelerated the catalytic process was further evaluated. Interestingly, a supplementation of of AMY1 and enhanced the amylase activity. glycine obviously enhanced the activity at both pH 8.0 and Due to the special habitats of Alicyclobacillus strains 9.0, while excessive glycine remarkably inhibited the cat- and the abundant production of hydrolases, isolation of alytic activity of AMY1. As shown in Fig. 4b, the highest thermophilic enzymes from these strains received much activity was detected in the 10 mM glycine/NaOH buff- attention in recent years. Most of Alicyclobacillus hydro- ered systems of pH 8.0 or 9.0 while decreased activities lases were found to be thermophilic, thermostable, and were observed upon increased glycine additions. acidophilic (optimal pH 3.0–6.0), except the xylanase Moreover, AMY1 was very stable after incubating in both from Alicyclobacillus sp. A4. This xylanase showed max- pH buffers of citrate/Na2HPO4 (pH 6.0) and glycine/ imum activity at pH 7.0 and displayed more than 40% NaOH (pH 9.0) for 120 min, with around 80% residual activity in a pH range of 3.8–9.4(Baietal.2010). In this activity detected at both mixtures (Fig. 4c). The influence study, by using different buffer solutions, it was firstly of glycine on thermal stabilization of proteins and en- disclosed that such Alicyclobacillus α-amylase also pos- zymes are well documented (Santoro et al. 1992;Nath sessed high activity under alkaline conditions of glycine- and Rao 2001; Goller and Galinski 1999). They suspected buffered. This finding may broaden the application

Fig. 4 Effect of pH on AMY1 activity and stability. a pH profile of activities of AMY1 after being pre-incubated in pH 6.0 or pH 9.0 AMY1 in different buffers; b activities of AMY1 in glycine/NaOH solutions for various times. The observed maximal activity was defined solutions (pH 8.0 and 9.0) with different glycine concentration; c as 100% Ann Microbiol (2018) 68:881–888 887

Fig. 5 Raw starch hydrolysis by the crude extract of AMY1. a In the citrate/Na2HPO4 solution of pH 6.0; b in the glycine/NaOH solution of pH 9.0. Hydrolysis was conducted at 65 °C for different time intervals and each substrate was hydrolyzed in triplicate

potentials of some Alicyclobacillus hydrolases and confers Conclusion versatility of these hydrolases in various industries, e.g., alkaline α-amylases have great potential in textile, deter- In this study, an Alicyclobacillusα-amylase was cloned and gent, food, and pharmaceutical industries. successfully expressed in E. coli BL21 CodonPlus with high expression level. Besides its acidic pH optima as those of Raw starch hydrolysis under acidic or alkaline other thermoacidiphilic Alicyclobacillus hydrolases, higher al- conditions kaline activity was found in the glycine-activated systems. This finding not only confers AMY1a broad pH range in utilizing starch, but also provides possibility for exploring In the course of conventional enzymatic saccharification, novel application fields of other Alicyclobacillus hydrolases. starch slurry is first gelatinizedbyheatinguptoatemper- ature of 105 °C and then subjected to two enzymatic — Funding This work was financial supported from the Key Scientific steps liquefaction and saccharification. As gelatinization Research Project of Shandong Province (2015GSF121020). increases the viscosity of the slurry, it poses a technical problem during mixing and pumping (Mamo and Compliance with ethical standards Gessesse 1999). The importance of enzymatic saccharifi- cation of raw starch without heating has become well rec- Conflict of interest The authors declare that they have no conflict of ognized in recent years (Sun et al. 2010). To test the ability interest. of AMY1 to digest raw starch, starch from potato, corn, sweet potato, and wheat was hydrolyzed using solutions of Research involving human participants and/or animals N/A pH 6.0 or pH 9.0. Different from the higher alkaline activ- Informed consent N/A ity toward soluble starch, relatively higher raw starch uti- lizing rates were observed at pH 6.0 (Fig. 5a) and slightly lower degrading efficiencies at pH 9.0 after 4-h incubation References (Fig. 5b). It is well known that soluble starch is formed from raw starch especially by relatively mild treatment Abe A, Tonozuka T, Sakano Y, Kamitori S (2004) Complex structures of with acids, by oxidation, or by heating with glycerol. We Thermoactinomyces vulgaris R-47 α-amylase 1 with suspected that the acidic environment contributes to the maltooligosaccharides demonstrate the role of domain N acting as – raw starch hydrolysis and thus gains a higher hydrolysis a starch-binding domain. J Mol Biol 335:811 822 Atlas RM (1993) In: Parks LC (ed) In: Handbook of microbiological efficiency at pH 6.0. Among tested raw starches, the media. CRC Press, Boca Raton, FL, p 843 highest hydrolysis efficiency was observed for potato Bai Y, Wang J, Zhang Z, Yang P, Shi P, Luo H, Meng K, Huang H, Yao B starch, with hydrolysis ratios of 50.4 and 45.9% observed (2010) A new xylanase from thermoacidophilic Alicyclobacillus sp. at pH 6.0 and pH 9.0, respectively. Hydrolysis ratio of A4 with broad-range pH activity and pH stability. J Ind Microbiol Biotechnol 37:187–194 wheat starch was also quite high, 42.9 and 39.7% at Boyce A, Walsh G (2015) Characterization of a novel thermostable pH 6.0 and 9.0, respectively. Differently, the utilization endoglucanase from Alicyclobacillus vulcanalis of potential appli- of corn starch was relative slow (30.8 and 24.2%, respec- cation in bioethanol production. Appl Microbiol Biotechnol 99: tively). The worst of all, the bigger granules and poor sol- 7515–7525 ubility of sweet potato starch resulted in very low hydro- CantarelBL,CoutinhoPM,RancurelC,BernardT,LombardV, Henrissat B (2009) The Carbohydrate-Active EnZymes database lysis rates; only around 13.0% of the substrate was hydro- (CAZy): an expert resource for glycogenomics. Nucleic Acids Res lyzed even after 4-h incubation. 37:233–238 888 Ann Microbiol (2018) 68:881–888

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