Identification of a Recombinant Inulin Fructotransferase (Difructose

Mar 20, 2015 - The recombinant enzyme showed maximal activity as 2391 units/mg at pH 6.5 and 55 °C. It displayed the highest thermostability among ...
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Identification of a Recombinant Inulin Fructotransferase (Difructose Dianhydride III Forming) from Arthrobacter sp. 161MFSha2.1 with High Specific Activity and Remarkable Thermostability Xiao Wang,† Shuhuai Yu,† Tao Zhang,† Bo Jiang,†,‡ and Wanmeng Mu*,†,‡ †

State Key Laboratory of Food Science and Technology, Ministry of Education, Key Laboratory of Carbohydrate Chemistry and Biotechnology, and ‡Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, People’s Republic of China S Supporting Information *

ABSTRACT: Difructose dianhydride III (DFA III) is a functional carbohydrate produced from inulin by inulin fructotransferase (IFTase, EC 4.2.2.18). In this work, an IFTase gene from Arthrobacter sp. 161MFSha2.1 was cloned and expressed in Escherachia coli. The recombinant enzyme was purified by metal affinity chromatography. It showed significant inulin hydrolysis activity, and the produced main product from inulin was determined as DFA III by nuclear magnetic resonance analysis. The molecular mass of the purified protein was calculated to be 43 and 125 kDa by sodium dodecyl sulfate−polyacrylamide gel electrophoresis and gel filtration, respectively, suggesting the native enzyme might be a homotrimer. The recombinant enzyme showed maximal activity as 2391 units/mg at pH 6.5 and 55 °C. It displayed the highest thermostability among previously reported IFTases (DFA III forming) and was stable up to 80 °C for 4 h of incubation. The smallest substrate was determined as nystose. The conversion ratio of inulin to DFA III reached 81% when 100 g/L inulin was catalyzed by 80 nM recombinant enzyme for 20 min at pH 6.5 and 55 °C. All of these data indicated that the IFTase (DFA III forming) from Arthrobacter sp. 161MFSha2.1 had great potential for industrial DFA III production. KEYWORDS: Arthrobacter sp., difructose dianhydride III, inulin, inulin fructotransferase, thermostability

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INTRODUCTION Inulin is a polymer of D-fructose molecules with a terminal Dglucose, in which the D-fructose units are linked together by β(1, 2) glycosidic linkage. It has been widely used as a soluble dietary fiber with many nutritional benefits within the food industry.1 Inulin is widely found in nature in a variety of plants, and it may be used as a cheap material to biologically produce high-value products for the food industry,2 such as inulin-type fructosyl oligosaccharides3 and high-fructose corn syrup.4 Recently, researchers pay increasing attention on bioconversion of inulin to difructose dianhydride III (α-D-fructofuranose-β-Dfructofuranose 2′,1:2,3′-dianhydride, DFA III), because DFA III has attracted considerable attention as a result of its low-calorie property and health benefits.5 DFA III has half sweetness but only 1/15 energy of sucrose,6 shows a promising prebiotic effect,7,8 and increases the absorption of minerals,9−11 flavonoids,12 and immunoglobulin G.13 Bioconversion of inulin to DFA III is catalyzed by inulin fructotransferase (IFTase) (DFA III forming) through inulin depolymerization and intramolecular fructosyl transfer.14 IFTase (DFA III forming) (EC 4.2.2.18) is a kind of extracellular microbial enzyme. The enzyme was first purified and identified from Arthrobacter ureafaciens15 and then was found widely existing in Arthrobacter species strains,16 such as Arthrobacter globiformis C11-1, 17,18 Arthrobacter ilicis OKU17B,19 Arthrobacter sp. H65-7,20 Arthrobacter sp. A-6,21 Arthrobacter sp. Buo141,22 Arthrobacter pascens T13-2,23 Arthrobacter sp. L68-1,24 Arthrobacter ureafaciens D13-3,25 and Arthrobacter aurescens SK 8.001.26,27 In addition, four non© 2015 American Chemical Society

Arthrobacter IFTases (DFA III forming) were characterized from Flavobacterium sp. LC-413,28 Bacillus sp. snu-7,29,30 Leifsonia sp. T88-4,31 and Nonomuraea sp. ID06A0189.32,33 The reported IFTases (DFA III forming) have more than 50% amino acid sequence identities with one another, but various source of IFTases (DFA III forming) display different enzymatic properties, especially the thermostability and specific activity.16 Thermostability and specific activity are two of the main factors contributing to the industrial application of IFTase (DFA III forming) for DFA III production. The higher specific activity that the biocatalyst has, the less amount of biocatalyst is required for bioconversion, and the better thermostability the IFTase (DFA III forming) exhibits, the higher conversion ability the enzyme displays during a long time of reaction. Several Arthrobacter strains of IFTases (DFA III forming) have been characterized having good thermostability, such as the strains from Arthrobacter sp. L68-1,24 Arthrobacter sp. A-6,21 and A. aurescens SK 8.001.26,27 Especially, the IFTase (DFA III forming) from Arthrobacter sp. L68-1 may retain more than half of the initial activity after incubated at 80 °C for 1 h.24 Among the reported IFTases (DFA III forming), the IFTase from Flavobacterium sp. LC-413 displays relatively low thermostability but the highest specific activity at 5728 units/mg;28 Received: Revised: Accepted: Published: 3509

December 19, 2014 March 4, 2015 March 20, 2015 March 20, 2015 DOI: 10.1021/jf506165n J. Agric. Food Chem. 2015, 63, 3509−3515

Article

Journal of Agricultural and Food Chemistry

Figure 1. SDS−PAGE and gel filtration analysis of the purified recombinant enzyme. (A) SDS−PAGE of the protein stained with Coomassie Blue. Lane 1, molecular mass marker proteins; lane 2, the purified recombinant protein by metal-affinity chromatography. (B) Determination of the molecular mass of native protein. The column was calibrated with thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (44 kDa) as reference proteins, and the native enzyme was eluted at a retention volume corresponding to 125 kDa (labeled by the red circle). Purification of Recombinant IFTase. All purification steps were conducted at 4 °C. The culture broth was centrifuged at 10000g for 20 min to remove cell pellets. The supernatant containing 6× histidinetagged recombinant proteins was directly loaded on Chelating Sepharose Fast Flow resin for metal-affinity chromatography using an Ä KTA purifier (GE Healthcare, Sweden). The purification was performed according to the protocol of the manufacturer (pET His Tag System, Novagen). The protein concentration was calculated using the Bradford method, and the molecular mass was assessed by both sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) and gel filtration. For SDS−PAGE, 12 and 5% (w/v) acrylamide were used for the separating and stacking gels, respectively, and Coomassie Brilliant Blue R250 was used for protein staining. For gel filtration, the native purified protein was loaded on a Superdex 200 10/300 GL gel filtration column (GE Healthcare, Sweden) and was eluted with 150 mM NaCl in 50 mM phosphate buffer (pH 6.5) at a flow rate of 0.5 mL min−1. Identification of the Reaction Product from Inulin. The reaction products from inulin by the recombinant enzyme were separated by high-performance liquid chromatography (HPLC, Agilent 1260 system, Santa Clara, CA) equipped with a refractive index detector and a preparative column, Carbomix H-NP5 (5 μm, 10 mm inner diameter × 300 mm, Sepax Technologies, Inc., Newark, DE). The column was eluted at 80 °C with a flow rate of 0.8 mL/min. The main product was collected and freeze-dried in the 4.5 L FreeZone freeze-dry system (Labconco Corp., Kansas City, MO). The freeze-dried sample (30 mg) was dissolved in 0.45 mL of D2O and analyzed by NMR using a Bruker Avance III 400 MHz Digital NMR spectrometer (Bruker, Karlsruhe, Germany). The NMR was operated at 400 MHz for 1H NMR, 13C NMR, and two-dimensional (2D) NMR spectra, including 2D 1H shift correlated spectroscopy (1H−1H COSY), 1H-detected heteronuclear single-quantum coherence (1H−13C HSQC), and 1H-detected heteronuclear multiple-bond correlation (1H−13C HMBC). The sodium 4,4-dimethyl-4-silapentane-1-sulfonate (DSS) was used as a standard to reference the chemical shifts. Enzyme Assay. The enzyme activity was determined by measuring the amount of produced DFA III from inulin. The reaction mixture was composed of 1 mL of solution containing phosphate buffer (pH 6.5), substrate inulin (10 g/L), and purified enzyme (6 nM). The reaction was conducted at 55 °C for 5 min and was stopped by boiling for 10 min. The generated DFA III was determined by HPLC [system, Agilent 1260; column, Asahipak NH2P-50-4E system, 4.6 mm inner diameter × 250 mm; column temperature, 25 °C; mobile phase, 65% (v/v) of acetonitrile; elution rate, 1 mL/min; and detector, refractive index detector]. One unit of enzyme activity was defined as the

however, the values of most IFTases (DFA III forming) are less than 2000 units/mg. In this work, a novel IFTase was characterized from Arthrobacter sp. 161MFSha2.1. The structure of depolymerized products from inulin was identified as DFA III by nuclear magnetic resonance (NMR) analysis. The enzymatic properties were studied in detail and were compared to those of other IFTases. The enzyme displays the highest thermostability and the second highest specific activity among the already characterized IFTases (DFA III forming), suggesting that it has great potential for industrial DFA III production.

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MATERIALS AND METHODS

Chemicals and Reagents. The sugar standards, including DFA III, 1-ketose (GF2), and nystose (GF3), were from Wako Pure Chemical Industries (Osaka, Japan). Inulin was purchased from BENEO-Orafti NV (Tienen, Belgium). Chelating Sepharose Fast Flow, the resin for affinity chromatography, was obtained from Amersham Biosciences (Arlington Heights, IL). All other chemicals used in this study were at least of analytical grade, obtained from Sigma (St. Louis, MO) and Sinopharm Chemical Reagent (Shanghai, China). Plasmids, Microorganisms, and Culture Conditions. The plasmid pET-22b(+) and E. coli BL21(DE3) were used as a vector and host to express the target gene, respectively. The E. coli strain was cultured in the Luria−Bertani (LB) medium. The LB medium used was composed of 5 g/L yeast extract, 10 g/L trypone, and 5 g/L NaCl (pH 7.5). Gene Cloning and Expression. The nucleotide and protein sequence information were obtained from the National Center for Biotechnology Information (NCBI), with accession numbers NZ_KB895786 and WP_018778058, respectively. The full length of the encoding gene was designed to contain an in-frame 6× histidinetag sequence before the stop codon and NdeI and XhoI restriction sites at the 5′ and 3′ termini. The designed DNA sequence was synthesized by Shanhai Generay Biotech Co., Ltd. (Shanghai, China) and was subcloned into the pET-22b(+) vector with the same restriction sites. The recombinant plasmid was then transformed into E. coli BL21(DE3) for expression. A single colony of transformant was inoculated in LB medium plus ampicillin (100 μg/mL). When the cells were grown to an optimal density (600 nm) of 0.6 at 37 °C, protein expression was initiated by the addition of isopropyl-β-D-1thiogalactopyranoside (IPTG) to a final concentration of 1 mM and the extracellular expression was performed at 28 °C for 72 h. 3510

DOI: 10.1021/jf506165n J. Agric. Food Chem. 2015, 63, 3509−3515

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Journal of Agricultural and Food Chemistry Table 1. Chemical Shifts in 1H and 13C NMR Spectra δC (ppm)a group

carbon atoms

main product

DFA III standard

α-Fru

1 2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′

66.36 106.30 83.90 78.09 82.85 63.33 61.69 104.06 84.59 74.98 81.73 63.81

66.36 106.30 83.90 78.09 82.85 63.33 61.69 104.06 84.95 74.98 81.73 63.81

β-Fru

a

δH (ppm)a

DFA III standard reported previously 66.2 106.3 83.9 78.1 82.9 63.3 61.7 104.2 84.5 75.0 81.8 63.8

35

main product

DFA III standard

3.68, 3.65

3.68, 3.65

4.15 3.87 3.69 3.89, 3.86 4.05, 4.02

4.15 3.87 3.69 3.89, 3.86 4.05, 4.02

4.09 4.65 4.31 3.85, 3.65

4.09 4.65 4.31 3.85, 3.65

Chemical shifts were determined relative to the internal standard DSS (δC = δH = 0.00 ppm).

Figure 2. Identification of the structure of the main product produced from inulin by the recombinant enzyme. (A) 1H−13C HMBC spectrum of the purified main product. Connectivities of α-D-fructofuranose (2, 3′) β-D-fructofuranose and β-D-fructofuranose (2′, 1) α-D-fructofuranose were deduced by the correlation between α-D-Fru-C-2 and β-D-Fru-H-3′ and that between β-D-Fru-C-2′ and α-D-Fru-H-1, respectively. (B) Chemical structure of the main product. amount of enzyme catalyzing the formation of 1 μmol of DFA III per minute at pH 6.5 and 55 °C. Effect of the pH and Temperature. The optimum pH and temperature for recombinant IFTase (DFA III forming) were determined by assaying the enzyme activity in 50 mM different buffers (acetate buffer, pH 4.0−5.5; phosphate buffer, pH 6.0−7.0; and Tris−HCl buffer, pH 7.5−8.0) at 55 °C or at different temperatures (30−80 °C) in 50 mM phosphate buffer (pH 6.5). The thermostability

was determined by calculating the residual activity of the enzyme, which was pre-incubated at 55, 60, 70, and 80 °C for 4 h. The initial activity without pre-incubation was taken as a control. Conversion of Inulin to DFA III by Recombinant IFTase. Production of DFA III from inulin was studied in 100 mL of reaction volume using 80 nM recombinant enzyme at pH 6.5 and 55 °C. Different initial inulin concentrations were tested, including 50, 100, and 200 g/L. 3511

DOI: 10.1021/jf506165n J. Agric. Food Chem. 2015, 63, 3509−3515

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Journal of Agricultural and Food Chemistry

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RESULTS

Heterologous Expression and Protein Purification. The IFTase-encoding gene from Arthrobacter sp. 161MFSha2.1 is heterologously expressed in E. coli with a C-terminal 6× histidine tag. After expression by IPTG induction, extracellular inulin depolymerization activity is significantly detected, indicating that the recombinant enzyme is successfully secreted to the culture media. The culture supernatant is collected and loaded into a nickel-affinity column for enzyme purification. The recombinant enzyme is purified to homogeneity, showing a single band of 43 kDa in SDS−PAGE (Figure 1A). The molecular weight of the native enzyme determined by gel filtration is calculated to be 125 kDa (Figure 1B). These data indicate that the recombinant enzyme is a homotrimeric protein. NMR Analysis of the Reaction Product. The 13C and 1H NMR spectra are shown in panels A and B of Figure S1 of the Supporting Information, respectively. The chemical shifts obtained from the spectra (shown in Table 1) are compared to the results of the DFA III standard. The 13C NMR spectrum (see Figure S1A of the Supporting Information) shows 12 carbons containing two sugar carbons (106.3 and 104.1 ppm) with large downfield shifts and four methylene oxylated carbons (66.36, 63.81, 63.33, and 61.69 ppm) with upfield shifts, indicating the existence of two hexoses. The two hexose moieties are assigned as α-D-fructofuranose and β-D-fructofuranose by 1H−1H COSY (see Figure S3A of the Supporting Information) and 1H−13C HSQC (see Figure S3B of the Supporting Information). As shown in Figure 2, the C2 of α-Dfructofuranose (δC, 106.30 ppm) and the C2′ of β-Dfructofuranose (δC, 106.30 ppm) have HMBC correlation with the H3′ of β-D-fructofuranose (δH, 106.30 ppm) and the H1 of α-D-fructofuranose (δH, 106.30 ppm), respectively, indicating the α-D-fructofuranose (2, 3′) β-D-fructofuranose and α-D-fructofuranose (2′, 1) β-D-fructofuranose connectivities, respectively. Therefore, the produced compound is determined as α-D-fructofuranose-β-D-fructofuranose 2′,1:2,3′-dianhydride (DFA III). Effect of the pH and Temperature on the Recombinant Enzyme. The purified recombinant IFTase (DFA III forming) from Arthrobacter sp. 161MFSha2.1 shows relatively high activity under acidic conditions and exhibits optimum activity at pH 6.5 in 50 mM phosphate buffer (Figure 3). Its relative activity is more than 80% of the maximal activity under the pH range of 5.0−6.5 and decreases sharply when pH is shifted to the alkaline side. The optimal temperature of the recombinant enzyme is determined to be 55 °C (Figure 4A). The relative activity is kept at more than 95% of the maximal activity at the temperatures between 50 and 60 °C but decreases obviously at other temperatures. Although the relative activity is sensitive to the temperature (Figure 4A), the enzyme displays remarkably thermal stability (Figure 4B). When incubated at 55, 60, 70, and 80 °C for 4 h, the enzyme still retains 91, 86, 77, and 51% initial activity. Reaction Product Analysis. The recombinant enzyme shows specific activity at 2391 units/mg at optimum reaction conditions (pH 6.5 and 55 °C). When the substrate inulin is depolymerized completely by using excess recombinant enzyme, the reaction mixtures analyzed by HPLC contain DFA III as the main product and some minor products composed of sucrose (GF), GF2, and GF3. In addition, the

Figure 3. Effect of the pH on the recombinant enzyme activity. The enzyme was mixed with various buffers (50 mM): acetate buffer (pH 4.0−5.5), phosphate buffer (pH 6.0−7.0), and Tris−HCl buffer (pH 7.5−8.0). The maximal enzyme activity was set as a reference value of 100%, and relative activity was expressed as a percentage of the maximal activity. Each point represents the mean (n = 3) ± standard deviation.

catalytic activities toward different types of fructosyl oligosaccharides, such as GF2 and GF3, are tested. The recombinant enzyme cannot act on GF2 but hydrolyzed GF3 to DFA III and GF, suggesting that the smallest substrate is GF3. Conversion of Inulin to DFA III by Recombinant IFTase. Under the optimum pH and temperature, biological production of DFA III is studied using 80 nM recombinant enzyme and various concentrations of substrate inulin. The conversion of inulin to DFA III reaches 88, 81, and 58% after reaction for 20 min, when the initial inulin concentration is set as 50, 100, and 200 g/L, respectively (Figure 5).

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DISCUSSION Thus far, there are two types of IFTases that have been identified, which depolymerize inulin to DFA III and DFA I (αD-fructofuranose-β-D-fructofuranose 2′,1:2,1′-dianhydride), respectively.16,34 Both types of IFTases have been mostly characterized from Arthrobacter species strains.16 The genomic sequence of Arthrobacter sp. 161MFSha2.1 has recently been released in the GenBank database (accession number NZ_ARGU01000000). In the genome, there is an annotated gene encoding a hypothetical protein (accession number WP_018778058) proposed as fructotransferase. The protein displays relatively high homology with both IFTase (DFA III forming) and IFTase (DFA I forming). In this work, the protein was heterologously expressed in E. coli, and the recombinant protein was purified and characterized. The purified recombinant enzyme displayed depolymerization activity toward inulin. The reaction product from inulin was purified and determined as DFA III by NMR analysis. Thus, the enzyme was identified as IFTase (DFA III forming). On the basis of the results of SDS−PAGE and gel filtration, the recombinant enzyme was suggested as a homotrimer. It was the same as the result of the crystal structure of Bacillus sp. IFTase (DFA III forming).14 Also, the IFTase (DFA III forming) from Arthrobacter sp. A-6 was a trimeric enzyme.21 However, other characterized IFTases were suggested as monomers or dimers. For examples, the IFTases from A. 3512

DOI: 10.1021/jf506165n J. Agric. Food Chem. 2015, 63, 3509−3515

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Journal of Agricultural and Food Chemistry

Figure 5. Production of DFA III from inulin by the recombinant enzyme. Three initial concentrations of inulin, 50, 100, and 200 g/L, were used for DFA III production.

from Arthrobacter sp. A-6 (1,195 units/mg)21 and the hereinreported Arthrobacter sp. 161MFSha2.1 (2391 units/mg). On the contrary, the non-Arthrobacter IFTases (DFA III forming) with high specific activity usually display low thermostability (Table 2). For examples, the IFTases (DFA III forming) from Nonomuraea sp. ID06A018916 and Leifsonia sp. T88-431 were completely inactivated after incubation at 80 °C for 20 min and at 70 °C for 30 min, respectively; the IFTase from Bacillus sp. snu-7 was rapidly inactivated at 70 °C with half-life of only 6.6 min;30 and the IFTase from Flavobacterium sp. LC-413 retained less than half of the initial activity when incubated above 75 °C for 30 min.28 In comparison, some Arthrobacter IFTases exhibited higher thermostability. The IFTases (DFA III forming) from A. aurescens SK 8.00127 and Arthrobacter sp. L68-124 were stable up to 70 and 80 °C, respectively, after 30 min of incubation, and the IFTase from Arthrobacter sp. A-6 retained more than half of the initial activity after 300 min of incubation at 70 °C.21 Herein, a novel thermostable IFTase (DFA III forming) was reported having the highest thermostability, which was stable up to 80 °C for 4 h of incubation. Therefore, IFTase (DFA III forming) from Arthrobacter sp. 161MFSha2.1 has great potential for industrial application for DFA III production, because of both high specific activity and significant thermostability. The smallest substrate was determined to be GF3. IFTases (DFA III forming) from A. aurescens SK 8.00126,27 and Bacillus sp. snu-729 displayed the smallest substrate as GF3 and fructofuranosyl nystose (GF4), respectively (Table 2). In this study, the recombinant enzyme degraded inulin to DFA III as a major product and small amounts of GF, GF2, and GF3. The result was the same as that of A. ureafaciens D13-3.25 In comparison, the IFTases (DFA III forming) from Arthrobacter sp. L68-1,24 A. pascens T13-2,23 Arthrobacter sp. A-6,21 and Leifsonia sp. T88-431 produced GF3 and GF4 as minor products. The IFTase from A. aurescens SK 8.001 produced small amounts of GF2, GF3, and GF4.26 The native crude IFTase from Nonomuraea sp. ID06A0189 could produce a very small amount of D-fructose,33 while D-fructose was not produced from inulin by the purified recombinant enzyme.32 The authors suggested that the Nonomuraea sp. ID06A0189 might also secrete other inulin-degrading enzymes to produce D-fructose.32

Figure 4. Effect of the temperature on the activity and stability of the recombinant enzyme. (A) Effect of the temperature on the enzyme activity. The maximal enzyme activity was set as a reference value of 100%, and relative activity was expressed as a percentage of the maximal activity. (B) Effect of the temperature on the enzyme stability. The effect on stability was studied by incubating the enzyme for 4 h at different temperatures, and the activity of the unincubated enzyme was taken as 100%. Each point represents the mean (n = 3) ± standard deviation.

aurescens SK 8.001,27 A. ureafaciens D13-3,25 Flavobacterium sp. LC-413,28 and A. globiformis C11-1 were monomers,17 and the IFTases from Leifsonia sp. T88-4,31 Arthrobacter sp. L68-1,24 A. pascens T13-2,23 Arthrobacter sp. H65-7,20 and A. ilicis OKU17B19 were dimers. The recombinant enzyme displayed maximal activity at pH 6.5 and 55 °C. The optimum pH and temperature were not much different from those of other reported IFTases (DFA III forming).16 However, the IFTase (DFA III forming) from Arthrobacter sp. 161MFSha2.1 exhibited very high specific activity and significant thermostability when compared to others (Table 2). The specific activity reached 2391 units/mg, which was only less than that of IFTase (DFA III forming) from Flavobacterium sp. LC-413 (5728 units/mg).28 Interestingly, the IFTases (DFA III forming) from non-Arthrobacter strains have relatively high specific activities (>1000 units/mg), except the IFTase from Leifsonia sp. T88-4 (644 units/mg).16 By comparison, the IFTases from Arthrobacter strains show specific activities below 1000 units/mg, except the IFTases 3513

DOI: 10.1021/jf506165n J. Agric. Food Chem. 2015, 63, 3509−3515

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Journal of Agricultural and Food Chemistry Table 2. Comparison of Biochemical Properties of Various Microbial IFTases (DFA III Forming) strain

native or recombinant

specific activity (units/mg)

Arthrobacter sp. 161MFSha2.1 A. aurescens SK 8.001 A. aurescens SK 8.001 A. ureafaciens D13-3 Arthrobacter sp. L68-1 A. pascens T13-2 Arthrobacter sp. A-6

recombinant recombinant native native native native native

2391 NRb 381 290 NR NR 1195

Arthrobacter sp. H65-7 A. ilicis OKU17B A. globiformis C11-1 A. globiformis C11-1 A. ureafaciens Nonomuraea sp. ID06A0189 Nonomuraea sp. ID06A0189

native native recombinant native native recombinant nativec

NR 853 227 294 162.4 1991 NR

80 °C; 240 min 70 °C; 60 min 70 °C; 60 min 70 °C; 30 min 80 °C; 60 min 75 °C; 20 min 70 °C; >300 min 80 °C;