Low-temperature-active and salt-tolerant β-mannanase from a newly isolated Enterobacter sp. strain N18

Low-temperature-active and salt-tolerant β-mannanase from a newly isolated Enterobacter sp. strain N18

Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e7, 2015 www.elsevier.com/locate/jbiosc Low-temperature-active and salt-tolerant b-mannanas...

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Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e7, 2015 www.elsevier.com/locate/jbiosc

Low-temperature-active and salt-tolerant b-mannanase from a newly isolated Enterobacter sp. strain N18 Jia You,1 Jin-Feng Liu,1 Shi-Zhong Yang,1 and Bo-Zhong Mu1, 2, * State Key Laboratory of Bioreactor Engineering and Institute of Applied Chemistry, East China University of Science and Technology, Shanghai 200237, PR China1 and Collaborative Innovation Center for Biomanufacturing Technology, Shanghai 200237, PR China2 Received 23 April 2015; accepted 6 June 2015 Available online xxx

A low-temperature-active and salt-tolerant b-mannanase produced by a novel mannanase-producer, Enterobacter sp. strain N18, was isolated, purified and then evaluated for its potential application as a gel-breaker in relation to viscosity reduction of guar-based hydraulic fracturing fluids used in oil field. The enzyme could lower the viscosity of guar gum solution by more than 95% within 10 min. The purified b-mannanase with molecular mass of 90 kDa displayed high activity in a broad range of pH and temperature: more than 70% of activity was retained in the pH range of 3.0e8.0 with the optimal pH 7.5, about 50% activity at 20 C with the optimal temperature 50 C. Furthermore, the enzyme retained >70% activity in the presence of 0.5e4.0 M NaCl. These properties implied that the enzyme from strain N18 had potential for serving as a gel-breaker for low temperature oil wells and other industrial fields, where chemical gel breakers were inactive due to low temperature. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Enterobacter; b-Mannanase; Low-temperature-active; Salt-tolerant; Viscosity]

b-Mannanases, the main composition of hemicellulose, play an important role in hydrolyzing mannan (1). They belong to glycoside hydrolase family 5, 26 and 133 (2,3), which are active in randomly cleaving the b-1,4-glycosidic linkage in mannan, glucomannan, galactomannan, and galactoglucomannan to produce monosaccharides and oligosaccharides such as mannobiose and mannotriose (4). b-Mannanases have shown good application prospect in bleaching of pulps, scouring and desizing (5), improving the shortage of enzyme in animal digestive system (6), hydrolyzing polysaccharides to produce functional oligosaccharides for substitution of cane sugar, improvement of oxidation resistance and reinforce of immunity (6e8), and so on. The feature of breaking main chain to produce monosaccharides and oligosaccharides with low viscosity made b-mannanases have great potential in acting as gel-breakers. Galactomannan-based guar gum came from plants as a major constituent of hemicellulose, was widely planted in Asia. As typical thickening agents in oilfield, the low prices of raw materials have attracted a lot of attention. Up to date, hydraulic fracturing technology in which the high pressure pump was utilized to inject a liquid to crack the formation was the most effectual measure to improve low permeability well (9). The hydraulic liquid had to carry proppant (such as sands) to prop the opened cracks, but sands could not be suspended in water, so that highly viscous watersoluble polymers like guar gum was added in applications (9,10). * Corresponding author at: State Key Laboratory of Bioreactor Engineering and Institute of Applied Chemistry, East China University of Science and Technology, Shanghai 200237, PR China. Tel.: þ86 21 64252063; fax: þ86 21 64252485. E-mail address: [email protected] (B.-Z. Mu).

In order to make the oil or gas flow to the wellbore in well stimulation process, the hydraulic fluids had to reduce the viscosity by gel breakers and then be pumped out. Traditional chemical breakers were oxidizing agents, such as potassium persulfate and ammonium persulfate. However, oxidization was severely affected by environmental temperature especially low temperature. It was observed that chemical breakers display barely activity below 50 C. Moreover, chemical breakers could react with any reactant including tubing and hydrocarbons and the oxidized products might cause environmental pollution. The lack of specificity also made them easily consumed so that they might be exhausted before reaching the destination. Due to the drawbacks of traditional chemical breakers in low temperatures, b-mannanases as enzymatic gel breakers with environmentally friendly characteristics and high specificity have drawn more and more attention. They have been applied to reduce viscosity in oil exploitation to serve as gel breakers. McCutchen et al. (10) firstly reported a thermostable breaker from Thermotoga neapolitana 5068 which can hydrolyze guar gum to improve well stimulation and oil and gas recovery. However, few reports were about low-temperature-active and salt-tolerant mannanases. Moreover, low-temperature-active enzymes are valuable for various industrial fields where low temperatures are required or energy conservation need to be considered. In our research, an efficient b-mannanase-producer Enterobacter sp. strain N18 was obtained from soil samples. The bmannanase from strain N18 was characterized as a lowtemperature-active and salt-tolerant enzyme. It exhibited high activity in a broad range of pH with remarkable ability to decrease viscosity as well.

1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.06.001

Please cite this article in press as: You, J., et al., Low-temperature-active and salt-tolerant b-mannanase from a newly isolated Enterobacter sp. strain N18, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.06.001




Materials The substrates including guar gum, locust bean gum, xanthan gum and sodium carboxymethyl cellulose were purchased from SigmaeAldrich Co. (St. Louis, MO, USA). The AxyPrep Bacterial Genomic DNA Miniprep kit was purchased from Axygen Biosciences Inc. (San Diego, CA, USA). The HiTrap DEAE Sepharose FF column was purchased from GE Healthcare Co. (Fairfield, CT, USA). Modified guar gum, clean up additive, clay stabilizer, and cross-linking agent were provided by Shengli Oil Field (Dongying, Shandong, China). All other chemicals were of analytical grade. 16S rRNA gene universal primers for identification was 8F:50 AGAGTTTGATCCTGGCTCAG-30 and 805R:50 -GACTACCAGGGTATCTAATC-30 (11). The trace element used in media contained 0.03% MnSO4$H2O, 0.01% Na2MoO4$2H2O, 0.20% FeSO4$7H2O, 0.006% CuSO4 and 0.10% CaCl2. The formation water from daqing oil field was consisted of 112.7 mg/L CaCl2, 42.8 mg/L MgCl2, 1597.1 mg/L NaCl, 17.0 mg/L Na2SO4, 381.6 mg/L Na2CO3 and 3176.0 mg/L NaHCO3, and the total dissolved substance was 5327.2 mg/L. Screening and identification of mannanase-producing bacteria Soil samples from different environments were inoculated in 5% TSB (Tryptone Soya Broth) medium to incubate at 37 C for 72 h. Then the resulting broth was inoculated into a selective medium containing 1.4% (w/v) guar gum, 0.25% KH2PO4, 1% Na2HPO4$12H2O, 0.1% NH4NO3 and 0.01% MgSO4 and incubated at 37 C for 72 h. This step was repeated for 10 times, then the resulted broth was spread on LB plates and incubated at 37 C for 36 h. Inoculating the colonies appeared on the plates into 96 wells microplate with 200 mL media per well and incubated at 37 C for 48 h. The medium (GG) contained 1.0% (w/v) Guar gum, 0.25% KH2PO4, 1% Na2HPO4$12H2O, 0.1% NH4NO3, 0.01% MgSO4 and 1.0% (v/v) trace elements. After incubation, the broth was centrifuged (Sigma, Germany) at 4 C for 20 min to remove cells. The supernatant was firstly subjected to enzyme activity assay, and the corresponding bacteria with high activity were evaluated with viscosity assay. The colonies with high activities and good abilities in reducing viscosity were purified through streaking on fresh agar plates, subjected to identification and stored at 80 C with 25% (v/v) glycerol. The sequence of isolated strains was identified with automated ABI 3730 sequencer (Dye-Terminator Cycle Sequencing; Applied Biosystems) after genomic DNA extraction and PCR amplification. Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nih.gov/BLAST) was used to compare the sequence with homologous strains in GenBank. Enzyme activity analysis The enzyme activity was detected by DNS (3,5dinitrosalicylic acid) method at 50 C with 0.5% of guar gum in 20 mM phosphate buffer (pH 7.5) as substrate solution (12). Microplate Reader (M5, Molecular Devices, US) was utilized to detect absorbance at 550 nm (OD550). The amount of enzyme that released 1 mmol of mannose within 1 min was defined as one unit of mannanase activity (U/mL). To evaluate the ability in reducing viscosity, the enzyme was added into guar gum solution (1.4% guar gum in 20 mM phosphate buffer, pH 7.5), and stood at 40 C. The viscosity was determined at different time intervals. The percentage of the viscosity reduction in 10 min was defined as viscosity-reduced efficiency. The enzyme activity was assayed by following the procedure unless otherwise noted. Inactivated enzyme was used as blank control during analysis. Each test was repeated for 3 times. Selection of cultural conditions Konjac powder, LBG (locust bean gum), Dmannose, D-galactose, maltose, glucose, cane sugar, soluble starch and dextrin were used as the sole carbon source to replace guar gum in GG medium, respectively. To confirm the best carbon source, the cell-free broth was utilized to detect enzyme activity and viscosity reduction ability. Different concentrations of the best carbon source ranging from 0.1% to 3.0% were utilized to determine the optimal concentration. Then, 0.1% of NH4NO3, NaNO3, NH4Cl, tryptone, yeast extract or urea was used as the nitrogen source. 0.05e0.5% of the best nitrogen source were used to determine the optimal mass concentration. After optimization of carbon and nitrogen sources, enzyme activity and viscosity assay were conducted after incubating for 12, 18, 24, 36, 48, 60, 72, 84 and 96 h to determine the optimum fermentation time.

Characterization of the b-mannanase The optimal temperature was measured by incubating the reaction mixtures at 0e100 C at 10 C intervals. The optimal pH was determined over a pH range of 3.0e12.0. The buffer systems were as follows (each 50 mM): NaH2PO4 (pH 3.0e4.0), Na2HPO4eNaH2PO4 (pH 5.0e8.0), and Na2HPO4 (pH 9.0e12.0). To assess the thermal stability, the enzyme was preincubated at temperature between 50e70 C under the optimal pH for 5e60 min. For pH stability, the enzyme was pre-incubated at different pH values at 50 C for 4 h without substrates. The influence of metal ions and various reagents on the enzyme activity were determined in Na2HPO4eNaH2PO4 buffer (pH 7.5) at 50 C, 1 mM (final concentration) of Naþ, Kþ, Ca2þ, Co2þ, Ni2þ, Cu2þ, Mg2þ, Fe2þ, Fe3þ, Mn2þ, Zn2þ, Hg2þ, SDS, EDTA and 10% ethanol (v/v) was added in the reaction solution, individually. The effect of 0e4.5 M NaCl on the enzyme activity was determined at pH 7.5 and 50 C. Stability of the enzyme in NaCl and the formation water was also measured. The enzyme was pre-incubated at 50 C with 1 M, 2 M of NaCl (pH 7.5) or the formation water for 4 h, and then the residual enzyme activity was determined. To measure the purified enzyme activity against different substrates, 0.5% guar gum, konjac powder, LBG, xanthan gum and sodium carboxymethyl cellulose (CMC) in 20 mM pH 7.5 sodium phosphate buffer was used as the substrate solution. The kinetic parameters, Km and Vmax of the enzyme were determined by Lineweaver-Burk plot under optimal conditions. The concentrations of LBG and guar gum ranging from 2.0 to 10.0 mg/mL. Analysis of hydrolysis products and gel breaking performance of the bmannanase Hydrolysis of 1.4% of guar gum (w/v) by 3 U of purified b-mannanase was performed in 1 mL of 20 mM phosphate buffer, pH 7.5 at 40 C for 30 min, 1.0 h, 3 h and 24 h, and then the hydrolysis products were analyzed using thin layer chromatography (TLC). TLC was carried out on a silica gel plate twice using npropanol: ethanol: water (7: 1: 2, v/v) as the developing solvent. Then the plates were heated at 130 C after spraying with a mixture of methanol: sulfuric acid (95: 5, v/v). A mixture of mannose (M1), mannobiose (M2), mannotriose (M3) and mannotetraose (M4) were used as the standard. 1 mL of cell-free broth from Enterobacter sp. strain N18 was added in 100 mL of fracturing fluid which containing 0.35% modified guar gum, 0.15% clean up additive, 0.18% Na2CO3, 0.036% NaHCO3, 1.0% clay stabilizer, and 13.7% cross-linking agent. The viscosity of the fracturing fluid was detected after incubating for 3 h at 40 C. Inactivated enzyme was used as blank control during analysis. Nucleotide sequence accession number The nucleotide sequence for the Enterobacter sp. N18 16S rRNA was deposited in GenBank under the accession number KJ510421.

RESULTS Newly isolated b-mannanase producers After selective isolation, 737 bacterial isolates were cultured in the microplates. Four of them showed good ability in reducing viscosity (Fig. 1). The mannanase produced by strain N18 showed the highest activity and the greatest ability in reducing viscosity. As shown in Fig. 1, the crude mannanase from strain N18 could reduce the viscosity of guar gum solution from more than 800 mPa s to 100 mPa s in 2 h. The strain N18 was identified as Enterobacter (KJ510421) by

Zymograms The activity and the molecular mass of the unpurified enzyme from Enterobacter sp. N18 were detected in polyacrylamide gels containing the substrates. The zymograms were obtained by adding 0.2% locust bean gum in the 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (13,14). After electrophoresis, the gel was soaked in 2.5% Triton X100 with gentle shaking overnight at 4 C to remove SDS and re-fold the protein. Then the gel was incubated at 50 C for 1 h after washing by 50 mM PBS buffer (pH 7.0) for 3 times at 4 C. At last, the gel was stained with 1% Congo red, and destained with 1 M NaCl. Purification of b-mannanase The cell-free broth was subjected to acetone sulfate fractionation. The precipitated crude enzyme was dialyzed against 50 mM TriseHCl buffer (pH 7.5, buffer A) overnight and concentrated by polyethylene glycol. The resulting sample was loaded onto a HiTrap DEAE Sepharose FF column pre-equilibrated with buffer A, followed by a linear gradient of 0e1.0 M of NaCl. The protein concentration was determined by the Bradford method (15), using bovine serum albumin as the standard for calibration. The purified protein was subjected to 12% SDS-PAGE described by Laemmli (16) to determinate the molecular weight and then the sample was stored at 20 C.

FIG. 1. Characterization of viscosity reduction: four of strains with relatively high activity were subjected to viscosity-reduction assay. The crude enzyme from strain N18 can reduce viscosity of guar gum much faster than that of the other three ones.

Please cite this article in press as: You, J., et al., Low-temperature-active and salt-tolerant b-mannanase from a newly isolated Enterobacter sp. strain N18, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.06.001

VOL. xx, 2015 16S rRNA gene sequence analysis, and it exhibited 100% similarity with Enterobacter cloacae P101 (CP006580.1). Except for strain N18, the isolates N14, B24-1 and B24-2 were designated as Bacillus, Enterobacter and Pseudomonas (KJ510420), and exhibited maximum similarity with 16S rRNA gene sequence from Bacillus cereus strain cifa chp56 (KC895909.1, 100% sequence similarity), Enterobacter sp. AN43 (JN886721.1, 100% sequence similarity) and Pseudomonas putida strain Zn-2 (JX441333.1, 100% sequence similarity). Optimization of fermentative conditions As the most efficient mannanase producer, Enterobacter sp. strain N18 was subjected to optimal assays. As shown in Fig. 2C, konjac powder was



most beneficial for the production of b-mannanase. The mannanase activity when using konjac powder as carbon source was 10 times higher than using dextrin. In the meantime, the viscosity assay showed that the rate of reducing viscosity of guar gum solution was much faster when using konjac powder as the carbon source than others (Fig. 2A). In this study, 1% was determined as the optimal concentration of konjac powder (Fig. 2E). The DNS method indicated that there were no distinct differences between inorganic and organic nitrogen sources (Fig. 2D). However, the result of viscosity reduction assay showed remarkable differences between different nitrogen sources (Fig. 2B). The viscosity-reduction efficiency when using NH4NO3 as nitrogen source was 2 times faster than using yeast extract.

FIG. 2. Effect of carbon sources, nitrogen sources and fermentative time on b-mannanase production: (A) The viscosity alterations of guar gum solution. The strain was fermented with different carbon sources. (B) The viscosity alterations of guar gum solution. The strain was fermented with different nitrogen sources. (C) Enzyme activities of b-mannanase from strain N18 grown in different carbon sources. (D) Enzyme activities of b-mannanase from strain N18 grown in different nitrogen sources. (E) Enzyme activities of b-mannanase from strain N18 grown in different concentrations of konjac powder. (F) Enzyme activities of b-mannanase from strain N18 grown in different concentrations of NH4NO3. (G) The viscosity alterations of guar gum solution. The strain was fermented with different time. (H) Enzyme activities of b-mannanase from strain N18 which was fermented with different time, data reflect the mean  SD (n¼3).

Please cite this article in press as: You, J., et al., Low-temperature-active and salt-tolerant b-mannanase from a newly isolated Enterobacter sp. strain N18, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.06.001



Most of the reported bacteria or fungi showed great differences in enzyme activity when fermented with different nitrogen sources such as Streptomyces with soybean meal (17) and Aspergillus sojae with beef extract (18). After optimization, activity of b-mannanase from strain N18 was nearly three times higher than before, and the viscosity reduction reached 95%. The optimal fermentation time was 60 h. The optimal carbon and nitrogen source were 1% of konjac powder and 0.1% of NH4NO3, respectively. Molecular mass, substrate specificity and kinetic parameters of the b-mannanase As shown in Fig. 3A, SDS-PAGE analysis of the purified b-mannanase displayed one single band with an apparent molecular mass of 90 kDa. The result was in agreement with the crude supernatants in the zymogram analysis (Fig. 3B). The purified mannanase exhibited specific activity of 3962 U/mg which was 10.94-fold higher than the unpurified one against guar gum (Supplementary Table S1). The activity of the b-mannanase from strain N18 with different substrate solutions showed that the enzyme had the highest activity of 8132  39 U/mg against LBG (100%). The mannanase activity against guar gum and konjac powder were 3962  21 U/mg (48.7%) and 7490  21 U/mg (92.1%), respectively. The enzyme showed barely activity against xanthan gum (3.0%), CMC (0), Pectin(0), Soluble starch(0) and xylan(0). The Km and Vmax values for the purified b-mannanase on LBG and guar gum were 3.427 mg/mL and 134.05 mmol mg1$min1 (Supplementary Fig. S1A) and 7.179 mg/mL and 97.37 mmol mg1$min1 (Supplementary Fig. S1B), respectively. Higher Km value of guar gum than LBG suggested the higher affinity of LBG to the purified b-mannanase, which was in accordance with the result of substrate specificity. Performances of the b-mannanase The b-mannanase showed over 75% activity in the temperature of 30e60 C with the optimal temperature at 50 C (pH 7.0), and more than 40% of activity was retained at 70 C (Fig. 4A). It was worth noting that nearly 50% of activity was remained at 20 C which was 1998 U/mg, and even more than 30% was remained at 0 C which was 1213 U/mg against guar gum. After incubating the protein at 50 C and 60 C for 1 h, 95% and 77% of activity were retained, respectively (Fig. 4C).

FIG. 3. SDS-PAGE and zymogram analysis of the b-mannanase from strain N18. Lane 1, the crude enzyme precipitated by acetone; lane 2, the purified protein; lane 3, concentrated cell-free broth.

J. BIOSCI. BIOENG., The optimal pH of the b-mannanase was 7.5 (50 C) and over 70% activity was retained at the pH range from 3.0 to 8.0 (Fig. 4B). Meanwhile, after incubating for 4 h at 50 C with different pH, more than 60% of activity was retained between pH 3.0 and 10.0 (Fig. 4D). As shown in Table 2, the activity of b-mannanase was effected at different degrees compared to the sample without reagents added. Among the tests, enzyme activity was strongly inhibited by 1 mM Hg2þ, and partially inhibited by 1 mM Ca2þ, Ni2þ, Cu2þ, Fe3þ, Zn2þ, EDTA and 10% ethanol (v/v). 1 mM Co2þ, Fe2þ and SDS enhanced the activity of the b-mannanase for about 1.4-, 1.1- and 1.1-fold, respectively. Naþ, Kþ, Mg2þ, Mn2þ had little or no effect on the activity. It was interesting that the enzyme activity was not decreased in the presence of SDS. Moreover, the b-mannanase showed more than 70% of enzyme activity in the presence of 0.5e4.0 M Naþ (Fig. 4E). The enzyme displayed nearly 100% of initial activity after incubated with 1 M NaCl for 4 h. There was 70% and 40% of activity remained after incubating with 2 M NaCl and the formation water, respectively (Fig. 4F). Hydrolysis product analysis and gel breaking performance of the b-mannanase According to the TLC analysis of the hydrolyzed products (Supplementary Fig. S2), the appearance of high molecular mass oligosaccharide after 1 h, mannotetraose and mannotriose after 3 h and 24 h following the hydrolysis of guar gum indicated that the b-mannanase from strain N18 was indeed an endo-b-mannanase acting on the b-1,4-linkage inside mannan. The viscosity of the fracturing fluid was higher than 1000 mPa s after cross-linking. With the addition of enzyme from strain N18 for 3 h at 40 C, the viscosity of the fracturing fluid was reduced to less than 5 mPa s which meet the national and industrial standards. DISCUSSION In the selective isolation processes, the viscosity of selective medium was reduced rapidly. This phenomenon revealed the existence of mannanase-producers. Mixed soils instead of singlesourced soil were chosen for the screening in this study. Several different soils from different sources including humus, compost, and wetland soil were mixed evenly. In this way, selectivity for bacteria was kept to a maximum level so that as many genera as possible can be isolated. Although the traditional screening method is time-consuming and tedious, there is no doubt that screening is still the basic step in scientific research. DNS method is the universal way to estimate mannanase activity. The ability to reduce viscosity is also an important evaluating index for mannanase. Above all, in this study, a high-throughput method was utilized to screen high-active mannanase producer. Both of the viscosityreducing and enzyme activity were considered in the process of screening. Numbers of b-mannanase producers have been reported, including bacteria, fungi, plants and animals. The main origin of bmannanases were microorganisms, including Bacillus (19), Vibrio (20), Yeast (21), and so on. They could secrete mannanase to decompose mannan for their energy need. From another point of view, the information on ecology, epidemiology and pathogenicity of Enterobacter was much more frequently reported than that on enzyme and metabolic production. To the best of our knowledge, members of the genus Enterobacter had not been reported for mannanase production. The discovery of Enterobacter-produced mannanase helps us to enlarge the family of mannanaseproducers and expand the recognition of metabolites of the genus of Enterobacter. b-Mannanase is an inducible enzyme. The yield of enzyme could change along with the change of fermentation conditions. In this study, the substitution of guar gum in the medium by konjac

Please cite this article in press as: You, J., et al., Low-temperature-active and salt-tolerant b-mannanase from a newly isolated Enterobacter sp. strain N18, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.06.001

VOL. xx, 2015



FIG. 4. Characterization of the purified b-mannanase from strain N18. (A) Effect of temperature: the assay was performed at 0e100 C (pH 7.0). (B) Effect of pH: measured at 50 C with pH ranging from 2.0 to 12.0. (C) Thermostability: the enzyme was incubated at 50 C, 60 C and 70 C for 0e60 min before detecting the residual activity. (D) pH stability: the reminded activity was measured after incubating the enzyme without substrate at 50 C for 4 h in phosphate buffers of pH 2.0e12.0. (E) Effect of NaCl: to detect the activity, the enzyme was added in the substrate solution with different concentrations of NaCl. (F) Stability of enzyme in salt: the enzyme was preincubated with 1 M NaCl, 2 M NaCl or the formation water at 50 C for 0e4 h before detecting the residual activity. The activity of untreated enzyme was defined as 100%, data reflect the mean  SD (n¼3).

powder conduced to a maximum b-mannanase activity. A generally accepted mechanism for induction of mannanase is that the polysaccharide is partly hydrolyzed by hydrolases at first. Then the low molecular weight hydrolysate can easily enter the cell to notify the presence of substrate and stimulate the synthesis of enzymes (22). However, if easily metabolized sources (e.g., glucose) are used as the sole carbon source in the culture medium, the synthesis of mannanase appears to be controlled (23). As a result, lower level of mannanase was produced when mannose, glucose or galactose was

used as a sole carbon source. On the contrary, the mannanase activity is preferably induced by polysaccharides especially heterogeneous polysaccharides, containing mannose, glucose or galactose as monomeric units. Locust bean gum and guar gum (galactomannan mainly composed of mannose and galactose), and konjac powder (glucomannan mainly composed of mannose and galactose) significantly enhanced the b-mannanase production in strain N18. However, the enzyme activity did not be improved with

TABLE 2. The influence of reagents on the enzyme activity (n¼3). TABLE 1. Enzyme features of b-mannanases from different bacteria. Strain Enterobacter sp. N18 Aspergillus niger CBS513.88 Bacillus licheniformis Cryptopygus antarcticus Sphingomonas sp. JB13 Flavobacterium sp. Enterobacter sp. N18 Rhizomucor miehei Cellulosimicrobium sp. HY-13 Streptomyces sp. S27 Bacillus subtilis WY34 Paenibacillus cookii a b

Opt. temp. ( C)

Opt. pH

Specific activity (U/mg)


50 45 50e60 30 40 35 50 55 50 65 65 50

7.5 5.0 6.0e7.0 3.5 6.5 7.0 7.5 7.0 6.0 7.0 6.0 5.0

4099a w460a w790a w340a w68a w2a 3962b 1492b 967b 74b 888b 10b

This study 30 31 32 29 33 This study 34 25 26 27 28

Specific activity of purified enzyme against LBG at 20 C. Specific activity of purified enzyme against guar gum at optimal conditions.

Reagent None Naþ Kþ Ca2þ Co2þ Ni2þ Cu2þ Mg2þ Fe2þ Fe3þ Mn2þ Zn2þ Hg2þ SDS EDTA 10% Ethanol (v/v)

Relative activity (%) 100.0 101.4 95.2 86.4 136.8 83.4 85.7 96.4 107.0 85.7 92.3 83.0 23.8 114.3 69.6 79.1


1.65 1.61 2.59 4.06 1.98 3.29 1.97 0.91 2.10 1.16 2.73 1.82 4.88 0.78 1.62 4.61

Please cite this article in press as: You, J., et al., Low-temperature-active and salt-tolerant b-mannanase from a newly isolated Enterobacter sp. strain N18, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.06.001




the increase of the concentration of carbon source. Because the oxygen transfer might be inhibited if the original viscosity of the medium was too high. As a result, the growth of organisms would be limited (24). Although demand for b-mannanases is increasingly high, the reality of their applications has lagged behind their significance. The low activity and stability of mannanases increase the cost of the enzyme due to large-scale production. Indeed, some mannanases produced by bacteria such as the genera of Bacillus, Cellulosimicrobium and Streptomyces had specific activity as high as 2000 U/mg against LBG at optimal conditions. However, most bmannanases from wild bacteria exhibited low activity of less than 500 U/mg. As a catalyst, high specific activity always attract great attention in various industrial fields. Based on reports, the wild strains such as Cellulosimicrobium sp. strain HY-13 (25), Streptomyces sp. S27 (26) and Bacillus subtilis WY34 (27) can produce bmannanases with extraordinarily high specific activity. Their specific activities against LBG were 8498 U/mg, 2107 U/mg and 8302 U/mg, respectively. These data were relatively similar to the endo-b-mannanase from strain N18. It was worth noting that, compared with LBG, their specific activities were only less than 15% activity against guar gum (Table 1). In this study, the endo-bmannanase from strain N18 was nearly 50% activity. For most wild bacteria, like Paenibacillus cookie (28), the enzyme activity was much lower than that of Enterobacter sp. strain N18. This performance make the enzyme exhibit great potential as a gel-breaker in oil fields which use guar gum as thickening agents in fracturing fluid. Salt-tolerant and cold-active enzymes with a broad pH range exhibit great potential application values. Except for the complicated environment in oil fields, utilization of marine resources, such as aquaculture of marine products, also demand salt-tolerant enzymes by considering the high salinity. Cold-active character is required to remedy the limitation of traditional chemical gelbreakers. Moreover, the pH of digestive tract and the temperature of fish (or poikilotherm) need to be taken into account in feed industry. Up to date, only few cold-active b-mannanases had been reported, and they were three of GH26 endo-1,4-b-mannanases from Sphingomonas sp. JB13 (29), Aspergillus niger CBS513.88 (30) and Bacillus licheniformis (31), a cold-active GH5 endo-1,4-mannanase from Cryptopygus antarcticus (32), and a family-unidentified mannanase from Flavobacterium sp. (33). Compared with the other cold-active b-mannanases, the specific activity of endo-b-mannanase from strain N18 was higher (Table 1). In conclusion, we described a rather stable, cold-active and salttolerant extracellular endo-b-mannanase produced by a novel mannanase-producer Enterobacter sp. strain N18. The enzyme had high activity against guar gum, LBG and konjac powder. Moreover, it was able to make the viscosity of guar gum solution decreased by more than 95% within 10 min. The properties of its broad working temperature especially low temperature, salt-tolerance and wide working pH range suggest the potential of the enzyme as a good candidate in complicated environmental applications, especially low temperature processes. To meet the commercial demands, improvement of the yield by means of genetic engineering and mutation will be our future objective. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2015.06.001.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (31200101), the 863 Program (2013AA064403).

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Please cite this article in press as: You, J., et al., Low-temperature-active and salt-tolerant b-mannanase from a newly isolated Enterobacter sp. strain N18, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.06.001