Heterologous expression and biochemical characterization of a GHF9 endoglucanase from the termite Reticulitermes speratus in Pichia pastoris

Heterologous expression and biochemical characterization of a GHF9 endoglucanase from the termite... Background: Cellulases are of great significance for full utilization of lignocellulosic biomass. Termites have an efficient ability to degrade cellulose. Heterologous production of the termite-origin cellulases is the first step to realize their industrial applications. The use of P. pastoris for the expression of recombinant proteins has become popular. The endoglucanase from Reticulitermes speratus (RsEG), belonging to glycoside hydrolase family 9 (GHF9), has not been produced in P. pastoris yet. Results: A mutant RsEG (G91A/Y97W/K429A) was successfully overexpressed in P. pastoris. RsEG , with optimum m m pH 5.0, was active over the pH range of 4.0 to 9.0, and exhibited superior pH stability over between pH 4.0 and pH 11.0. It displayed the highest activity and good stability at 40 °C, but lost activity quickly at 50 °C. The apparent kinetic parameters of RsEG against Carboxymethyl Cellulose (CMC) were determined, with K and V of 7.6 mg/ m m max 2+ 2+ 2+ ml and 5.4 μmol/min� mg respectively. Co ,Mn and Fe enhanced the activity of RsEG by 32.0, 19.5 and 11.2% 2+ 2+ respectively, while Pb and Cu decreased its activity by 19.6 and 12.7% separately. Conclusions: RsEG could be overexpressed in P. pastoris. It was stable between pH 4.0 and pH 11.0, and exhibited higher stability at temperatures ≤ 40 °C. This endoglucanase may have potential to be used in the field of laundry, textile and lignocellulose-based biofuels and chemicals. Keywords: Reticulitermes speratus, GHF9 endoglucanse, Heterologous expression, Pichia pastoris, Enzymology Background degradation of cellulose is mainly performed by cellu- Lignocellulosic biomass obtained as agricultural and in- lases produced by microorganisms. To breakdown cellu- dustrial byproducts is an abundant, inexpensive and re- lose efficiently, three classes of cellulases are needed to newable source, and is a desirable feedstock for the work synergistically: endoglucanases (EGLs, EC 3.2.1.4), sustainable production of liquid fuels and chemicals cellobiohydrolases (CBHs, EC 3.2.1.91) and β-glucosidases through the biorefinery processes [1, 2]. Lignocellulose (BGLs, EC 3.2.1.21) [3, 4]. EGLs hydrolyze intramolecular is mainly composed of cellulose, hemicellulose and lig- β-1,4-glucosidic linkages in cellulose randomly, whereas nin, among which cellulose is the major polysaccharide. CBHs cleave cellulose from the reducing and non- The turnover of cellulose plays an important role in glo- reducing ends in a progressive process. BGLs degrade cel- bal carbon cycle for all living organisms. In nature, the lobiose into glucose. According to the CAZy (Carbohy- drate-Active enZYmes) database, where glycosidases are classified according to similarities in the protein sequence * Correspondence: lemomlyty@sohu.com; ygdu@ipe.ac.cn; jjli@ipe.ac.cn and three-dimensional structure, cellulases belong to Sichuan Normal University, College of Life Science, Chengdu 610101, China National Key Laboratory of Biochemical Engineering, National Engineering glycoside hydrolase families (GHF) 5 to 10, 12, 26, 44, 45, Research Center for Biotechnology (Beijing), Key Laboratory of 48, 51, 61 and 74, etc. [5]. Biopharmaceutical Production & Formulation Engineering, PLA, Institute of Process Engineering, Chinese Academy of Sciences, No. 1 North 2nd Street, Beijing 100190, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. BMC Biotechnology (2018) 18:35 Page 2 of 9 Termites (Isoptera or Termitoidae) are the main de- while symbiont cellulases work only in the hindgut [7, 8, graders in tropical and subtropical regions. They have a 15]. The roles of the endogenous cellulases played in the profoundly efficient ability to degrade cellulose [6–8], gut system are postulated to be as important as those and can digest 74 to 99% of the cellulose present in the their symbionts produced (mostly GHF5, GHF7 and plant material they ingest [9]. Thus termite guts are GHF45) in the hindgut for converting cellulose termites regarded as ‘the world smallest bioreactor’ [10, 11]. Ter- ingest [7, 8, 12]. mites are classified into higher- and lower ones based on Heterologous expression of the insect-origin cellulase the presence or absence of flagellated protistan symbi- genes is the first step to realize their industrial applica- onts in their hindguts [7, 8]. Many studies have been tions. Some insect GHF9 endoglucanases have been heter- performed in lower termites. This group of termites con- ologously overexpressed and biochemically characterized. tains a dual cellulose-degradation mechanism: endogen- For instance, RsEG was successfully overexpressed in E. ous cellulases and symbiotic cellulases degrade cellulose coli by using a directed evolution approach, and the ob- cooperatively [7, 8, 12, 13]. All endogenous EGLs exclu- tained mutant A18 was not only efficiently expressed in E. sively belong to the glycoside hydrolase family (GHF) 9 coli, but also showed a 20-fold higher activity than native [7–9, 13–15]. Cellulases of flagellate origin have also RsEG [28, 29]. Later, active RsEG and NtEG were also suc- been identified as members of GHF5, GHF7 and GHF45 cessfully obtained in Aspergillus oryzae [30]. Recombinant from hindgut flagellates of Coptotermes formosanus, C. CfEG3a, Cell-1, CfEG5a, CgEG1 and MbEG1 were also lacteus, Mastotermes darwiniensis and Reticulitermes produced in E. coli, and could hydrolyze cellulose [21–23, speratus [7]. CBHs are only found in the hindgut of 26, 27]. Moreover, active TeEG-I (baculovirus-infected in- lower termites [7, 16], whereas both EGLs and BGLs are sect Sf9 cells) [20], Cell-1 (baculovirus-infected insect Sf9 found in the midgut and hindgut of lower termites [11]. cells) [23], TcEG1 (Drosophila S2 cells and S. cerevisiae) In comparison, higher termites, which do not have fla- [24, 25], and MbEG1 (P. pastoris)were successfullyover- gellates in their hindguts, account for over 75% of ter- expressed in the eukaryotic expression systems [27]. So mite species. The cellulolytic systems of higher termites far, only the kinetic parameters of several insect-origin are different from those of lower ones. Studies have GHF9 endoglucanases were determined, including TeEG- demonstrated that the majority of cellulase activity of I, CfEG3a, RsEG, NtEG, Cell-1, CfEG5 and CgEG1 [20– higher termites takes place in the midgut, suggesting 23, 26, 30]. Eight biochemically characterized insect GHF9 that they mainly depend on endogenous cellulases for endocellulases including RsEG, NtEG, CfEG3a, CfEG5a, cellulose degradation [6, 7, 15]. Metagenomic analysis TcEG1, MbEG1, Cell-1 and TeEG-I were aligned, and the revealed a diverse set of genes related to glycoside hy- sequence identity between them is from 61.6 to 63.2% drolases in the hindgut of a higher termite Nasutitermes (Additional file 1). sp., implying that hindgut microbes also play an import- Reticulitermes speratus is one of the most-extensively ant role for cellulose degradation [17]. Proteome analysis investigated termites in terms of its cellulolytic systems of the bacterial community in a higher termite Nasuti- [7, 8, 14]. Glycoside hydrolases from Reticulitermes sper- termes corniger indicated that bacterial enzymes play atus, belonging to GHF3, GHF7, GHF9 and GHF45, more significant roles in metabolism than in activities were heterologously overexpressed in E. coli, S. crevisiae related to cellulose degradation [18]. and A. oryzae respectively [31, 32]. However, no glycosi- Since identification of an endogenous cellulase gene dases from Reticulitermes speratus have been produced (RsEG)in Reticulitermes speratus by Watanabe et al. in P. pastoris. So far, only one GHF9 endo-glucanase [14], encoding an EGL in GHF9, a lot of insect-origin from termite Macrotermes barneyi was successfully GHF9 cellulase genes have been cloned and/or analyzed, expressed in P. pastoris. Methylotrophic yeast P. pastoris such as NtEG from the higher termite Nasutitermes can strongly over-expresses foreign proteins and serve as takasagoensis [19], NwEG from Nasutitermes walker an expression system for insect proteins [33, 34]. There- [19], TeEG-I from the cricket Teleogryllus emma [20], fore, in this study, an RsEG mutant (G91A/Y97W/ CfEG3a and CfEG5 from Formosan subterranean termite K429A) named as RsEG was heterologously overexpro- (Coptotermes formosanus)[21, 22], Cell-1 from Reticuli- duced in P. pastoris, and recombined RsEGm was fully termes flavipes [23], TcEG1 from red flour beetle Tribo- characterized, including optimal pH and temperature, lium castaneum [24, 25], CgEG1 from the Brazaian pH and thermal stability, kinetic parameters and impact termite Coptotermes gestroi [26], MbEG1 from the of divalent metal ions on the enzymatic activity. fungus-growing higher termite Macrotermes barneyi [27], etc.. These cellulase-coding genes are predomin- Results antly expressed in salivary glands/midgut/foregut [7, 8, Overexpression of RsEG in P. pastoris 15, 23]. Salivary gland−/midgut-secreted endogenous The codon optimized gene RsEG encoding endogluca- cellulases may work in both the midgut and the hindgut nase from Reticulitermes speratus (GenBank: AB008778. Zhang et al. BMC Biotechnology (2018) 18:35 Page 3 of 9 2) with three mutations (G91A/Y97W/K429A) was syn- residual activities at pH 9.0 and pH 10.0 respectively, and thesized [29] (Additional file 2), and cloned into the ex- exhibited very low activity (< 15.0% residual activities) at pression vector pPICZαA at the restriction sites of EcoRI pH 3.0 and pH 11.0. Therefore, RsEG was active over and XbaI. The obtained construct pJL36 was confirmed the pH range of 4.0 to 9.0. Our result is slightly different by DNA sequencing. Linearized construct by BstXI was from the published one for recombinant WT RsEG pro- transformed into P. pastoris GS115 by electroporation. duced in A. oryzae: it had similar optimal pH (pH 5.5) to Nine transformants were randomly picked, grown and RsEG , but exhibited superior activity within a narrow used for PCR. The agarose gel electrophoresis results of pH range (pH 5.0 - pH 7.5) [30]. the PCR products of nine transformants, corresponding The pH stability of RsEG was investigated after being to the size (~1800 bp) of RsEG plus α-factor signal se- pre-incubated for a fixed time over the pH range of 3.0 quence, c-myc epitope and His tag, confirmed that the to 11.0 (Fig. 3). Notably, RsEG was stable over the pH 6 m RsEG gene was successfully inserted into genome P. pas- range of 4.0 to 11.0, retaining more than 74.0% of ori- toris GS115 (Fig. 1). ginal activity after 120 h. It was unstable at pH 3.0, and Five (pJL36A, pJL36C, pJL36E, pJL36G and pJL36I) out only kept 32.5% of initial activity after 120 h. RsEG ex- of nine transformants were used to screen the one with hibited good pH stability over a wide pH range (pH 4.0 - the highest protein expression level. As shown in pH 11.0). The results are very similar to the reported Additional file 3, pJL36A, pJL36C and pJL36E showed ones for recombinant WT RsEG in A. oryzae, in which higher protein production level (Additional file 1:Figure it retained over 80% initial activity after 20 h of pre- S1). According to the amino acid sequence of pJL36 and incubation between pH 5.0 and 10.0, and lost activity the C-terminal peptide containing c-myc epitope and His sharply at pH 3.0 [30]. tag, the predicted molecular weight of pJL36 is around 48. 7 kDa, which was much lower than the apparent molecu- Determination of optimal temperature and thermal lar weight on SDS-PAGE (Additional file 3). The differ- stability of RsEG ence between the predicted molecular weight and the The optimal temperature of RsEG was determined apparent one for pJL36 was possibly due to the fact that (Fig. 4). It showed the highest activity at 40 °C, and highly glycosylated proteins are usually obtained when kept > 72.8% of residual activity between 20 and 45 °C. they are overexpressed in P. pastoris [32]. After pJL36A, However, it lost activity rapidly when temperature rose pJl36C and pJL36E were further induced with 0.5% up to 50°C, retaining only 27.7% of original activity. It methanol for different time intervals, it was found maintained only 18.4% of residual activity at 65°C. Our pJL36C produced higher protein yield and used for sub- result is very similar to the published one for recombin- sequent large-scale overexpression (Additional file 4). ant WT RsEG in A. oryzae, where it had optimal activ- Overexpression of pJL36C was also confirmed by west- ity at 45°C, and lost activity sharply at temperatures ern blotting (Additional file 5) and CMC activity. above 50°C [30]. The thermal stability of RsEG was investigated after Determination of pH optima and pH stability of RsEG being pre-incubated for a fixed time at pH 5.0, and at Britton-Robinson (B & R) buffer is a “universal” buffer 30, 40 and 50 °C respectively (Fig. 5). RsEG was stable used for the range of pH 3.0 to pH 11.0, so it was chosen at 30°C, and lost only 5.5% of original activity after for determining optimal pH and pH stability of RsEG . 120 h of pre-incubation. It kept 66.0% of initial activity As shown in Fig. 2, RsEG showed the highest activity at 40°C after 120 h. It was completely inactivated at 50 ° at pH 5.0, and retained original activities above 77.6% be- C after 120 h, and retained only 15.1% of original activity tween pH 4.0 and pH 8.0. It maintained 66.3 and 48.0% at 50°C after 1 h. Therefore, RsEG was a thermolabile Fig. 1 Agarose gel for PCR products of nine randomly picked transformants. Lane 1: DNA ladder; Lanes 2-10: PCR products of 9 randomly picked transformants (pJL36A - pJL36I) Zhang et al. BMC Biotechnology (2018) 18:35 Page 4 of 9 Fig. 2 Effects of pH on RsEG activity. Values are expressed as the means of three replicates ± standard deviation. The activity of the enzyme at pH 5.0 and 37 °C (6.7 U/mg) was defined as 100% endocellulase, and was fairly stable below 30°C. The re- Effects of divalent metal ions on enzyme activity sults are very similar to the reported ones for recombin- The effects of divalent metal ions on RsEG activity were 2+ 2+ 2+ ant WT RsEG in A. oryzae, where it retained over 80% examined (Fig. 7). Co ,Mn and Fe upregulated the of maximum activity after 30 min-incubation at temper- cellulolytic activity of RsEG by 32.0, 19.5 and 11.2% 2+ 2+ atures as low as 45°C and was not stable at temperatures respectively, while Pb and Cu decreased the activity of above 50°C [30]. RsEG by 19.6 and 12.7% separately. Other divalent metal ions didn’t show obvious influence on the catalytic activity Determination of kinetic parameters of RsEG of RsEG . The kinetic parameters of recombinant RsEG against CMC were determined by substrate hydrolysis from 0.2 to Discussion 2% (w/v) for 5 min. The deduced kinetic values were ap- In the present study, a GHF9 endoglucanase RsEG mu- parent parameters since saturation was not achieved even tant (RsEG ) from Reticulitermes speratus was overex- when high CMC concentrations were used (Fig. 6). The pressed in P. pastoris and characterized. BLAST (Basic apparent K and V values of RsEG towards CMC were Local Alignment Search Tool) search of RsEG resulted m max 7.6 mg/ml and 5.4 μmol/min•mg respectively. in significant matches of GHF9 cellulases originating Fig. 3 pH stability of RsEG . The pH stability assay was investigated by first pre-incubating RsEG in 50 mM B & R buffer at different pH values m m (pH 4.0 to 11.0) at 4°C for 1 h, 5 h, 24 h, and 120 h respectively. The residual activities were then measured immediately under standard conditions (optimal pH, 37°C for 15 min). The initial activity at optimal pH (5.0) and 37°C (6.7 U/mg) was taken as 100%, and the percentage of the residual activity at different time points and pH values against the original one at optimal pH (5.0) was calculated. Values are expressed as the means of three replicates ± standard deviation Zhang et al. BMC Biotechnology (2018) 18:35 Page 5 of 9 Fig. 4 Effects of temperature on RsEG activity. Values are expressed as the means of three replicates ± standard deviation. The original activity at pH 5.0 and 40 °C was taken as 100% (7.9 U/mg) from insects. Among top 100 Blast Hits, only nine endo- were even expressed in the complicated baculovirus ex- cellulaseas were heterologously overexpressed and partly pression system [23, 24]. characterized. As far as we know, this is the first report In comparison with other insect GHF9 endo- on heterologous expression of active RsEG in P. pastoris. cellulaseas, RsEG exhibited higher specific activity than Until now, only one insect endo-cellulase belonging to Cell-1 (~1 U/mg) and comparable activity to crude GHF9, MbEG, was heterologously overexpressed in P. CfEG3a (14-19 U/mg) [21, 23], and much lower specific pastoris [27]. Though active WT RsEG was not pro- activity than other characterized insect GHF9 endoglu- duced in E. coli and S. cerevisiae, an active mutant A18 canases such as recombinant RsEG in A. oryzae (112 U/ was successfully obtained through DNA shuffling of four mg), CfEG5 (325 U/mg), NtEG (105 U/mg), MbEG1 orthologous parental cDNAs in E. coli [28]. Active re- (223.9 U/mg) and TeEG1 (948.1 U/mg) [20, 22, 27, 30]. combinant RsEG was also successfully overexpressed in The lower activity of RsEG recombined in P. pastoris A. oryzae [30]. So far, most insect GHF9 endoglucanases could be presumably due to the reason that RsEG pro- were produced in E. coli [21–23, 26, 27], and several duced in P. pastoris was not correctly folded and/or glycosylated. It appears that most termite GHF9 endoglucanases had optimal pH value around 5.0-6.0, including CfEG3a from Coptotermes formosanus (pH 5.0) [21], MbEG1 from Macrotermes barneyi (pH 5.5) [27], CfEG5 from Coptotermes formosanus (pH 5.6) [22], NtEG from Fig. 5 Thermal stability of RsEG .RsEG was pre-incubated for varied m m times (15 min to 2 h) at pH 5.0, and 30°C, 40°C and 50°C respectively, and samples were chilled on ice for at least 10 min. Afterwards the residual activities were measured under standard conditions (optimal pH, 37°C for 15 min). The original activity at pH 5.0 and 37°C (6.7 U/ mg) was taken as 100%, and the percentage of the residual activity at different time points and temperatures against the initial one was calculated. Values are expressed as the means of three replicates ± Fig. 6 Effects of CMC concentration on RsEG activity. Values are standard deviation expressed as the means of three replicates ± standard deviation Zhang et al. BMC Biotechnology (2018) 18:35 Page 6 of 9 Fig. 7 Effects of divalent metal ions on RsEG activity. The effects of metal ions on the catalytic activity of RsEG were determined by adding m m 1 mM of various divalent metals to the standard enzyme assay system (pH 5.0, 37°C for 15 min). The activity at pH 5.0 and 37°C (6.7 U/mg) in the absence of divalent metal ions (control) was taken as 100%, and the percentage of the activity in the presence of different divalent metal ions against the control was calculated. Values are expressed as the means of three replicates ± standard deviation Nasutitermes takasagoensis (pH 6.0) [30], and CgEG1 incubation at 50°C, and dramatically lost activity at tem- from Coptotermes gestroi (pH 6.0) [26]. The optimum peratures above 55°C [27]. The activity TeEG-I was stable pH of TeEG-I from the cricket Teleogryllus emma with up to 45°C during 10 min of incubation, but was lost after 61% sequence identity to RsEG, was determined to be 5. 10 min-incubation at 75°C [20]. The t values of CgEG1 1/2 0[20]. By contrast, recombinant TcEG1 from red flour at 45, 50, 60 and 70 °C were 46.21, 8.2, 1.48 and 0.35 min, beetle Tribolium castaneum showed the maximum ac- respectively [26]. NtEG retained 60% of maximum activity tivity at pH 12.0. Only one neutral insect-origin GHF9 after 30 min of pre-incubation at temperatures as low as endocellulase – Cell-11, from the termite Reticulitermes 60°C, and lost activity sharply above 60°C [30]. Thus, it flavipes, was identified and characterized, with optimal seems that NtEG and MbEG1 were thermally stable than pH between 6.5 and 7.5 [23]. Therefore, most biochem- RsEG, whereas it displayed comparable thermal stability ically characterized insect GHF9 endoglucanases were to TeEG-I and CgEG1 [20, 26, 27, 30]. classified as acidic cellulases. Comparison of the charac- In the case of the kinetic parameters of insect GHF9 terized insect GHF9 endoglucanases revealed that most endocellulases, the apparent K value of RsEG is com- m m of them were active over a wide pH range, including parable to that of Cell-1 expressed by BEVS (9.9 mg/ml) RsEG (pH 4.0 - pH 9.0), CfEG5 (pH 3.6 - pH 9.6), and in E. coli (14.7 mg/ml) [23], and is much higher than TeEG-I (pH 3.0 - pH 8.5) and Cell-1 (pH 4.0 - pH 9.0) those of WT RsEG expressed in A. oryzae (2.0 mg/ml), [20, 22, 23]. In contrast, MbEG1 (pH 4.5 - pH 6.5), CfEG3a (2.2 mg/ml), NtEG (4.7 mg/ml), and CfEG5 (5. TcEG1 (pH 8.0 - pH 12.0), and RsEG produced in A. 6 mg/ml) [21, 22, 30]. Therefore, it showed lower bind- oryzae showed higher activity over a narrow pH range ing affinity towards CMC. Although its V value was max (pH 5.0 - pH 7.5) [27, 30]. Moreover, RsEG exhibited much higher than those of Cell-1 produced by BEVS (1. higher thermal stability than recombinant WT NtEG in 06 μmol/min•mg) and in E. coli (0.84 μmol/min•mg) A. oryzae and MbEG, which retained over 75% of max- [23], it was greatly lower than those of WT RsEG imal activity after 30 min of incubation at pH 3.5 to expressed in A. oryzae (1429 U/mg) [30], CfEG3a pH 8.0 and was stable after 20 h of pre-incubation be- (590 U/mg) [21], NtEG (1667 U/mg) [30], and CfEG5 tween pH 5.5 and pH 9.0 respectively [27, 30]. (548 U/mg) [22]. Just like RsEG , TeEG-I, CfEG3a, CfEG5, MbEG1 and A lot of studies towards impact of divalent metal ions recombinant TcEG1 had highest enzymatic activity at on endo-cellulases were done [35]. However, until now, around 40°C [20–22, 25, 27]. The optimal temperatures there was only one report on insect-origin GHF9 endo- 2+ of CgEG1, and Cell1 were 50°C and in the range of 50- glucanases that Ca slightly enhanced CMC activity of 60°C respectively [23, 26]. Recombinant NtEG in A. ory- Cell-1, and stabilized its activity over time [23]. In our 2+ zae had optimal activity at 65°C [30]. Until now, only study, Ca didn’t show obvious impact on RsEG . the thermal stabilities of several insect GHF9 endogluca- Therefore, it seems that same metal ions had different nases were investigated. For example, MbEG1 retained influence on GHF9 endoglucanases from different 60% of its maximum activity after 30 min of pre- species of insects. Zhang et al. BMC Biotechnology (2018) 18:35 Page 7 of 9 Conclusions from agarose gels using the DNA gel extraction kit. Plas- In summary, a GHF9 endoglucanase RsEG mutant mid DNA was isolated using the plasmid miniprep kit. (RsEG ) from Reticulitermes speratus was successfully The plasmid for the synthesized RsEG gene with the m m recombined in P. pastoris and characterized in detail. restriction sites of EcoRI and XbaIat5′ and 3′-terminal Recombinant RsEG showed the highest activity at respectively, which was cloned into PUC57, was digested pH 5.0 and 40 °C, and was very stable between pH 4.0 with restriction enzymes EcoRI and XbaI, and re-cloned and pH 11.0. It exhibited higher stability at tempera- into the vector pPICZαA digested with EcoRI and XbaI, tures ≤ 40°C and was unstable above 45°C. The appar- respectively. The final construct was confirmed by DNA ent K and V values of RsEG against CMC were sequencing, and named as pJL36. m max m 2+ 7.6 mg/ml and 5.4 μmol/min•mg respectively. Co , 2+ 2+ Mn and Fe showed some stimulatory effects on Screening of recombinant colonies by direct PCR and 2+ 2+ RsEG ,while RsEG was inhibited by Pb and Cu . expression m m Therefore, RsEG exhibited good pH and thermal stability The construct pJL36 was linearized with BstXI and to an extent, and activities of insect endoglucanases may transformed into P. pastoris GS115 by electroporation be enhanced by some metal ions. following the manufacturer’s recommendation. Zeocin- resistant P. pastoris clones were grown on YPDS plates Methods containing 100 μg/ml Zeocin. 10-20 colonies were Materials picked and streaked for single colonies on fresh YPD or Chemicals were from Sigma, Merck or Ameresco. Oligonu- YPDS plates containing 100 μg/ml of Zeocin. cleotides and the codon-optimized RsEG gene encoding Nine transformants were randomly selected and grown an endo-β-1,4-glucanase from the termite Reticulitermes overnight in YPD. 10 μlof a Pichia pastoris culture was speratus with three mutations (G91A/Y97W/K429A) were placed into a 1.5 ml microcentrifuge tube, and 1 μlof synthesized by Shanghai Sangon Biotech Co. Ltd. (China) the culture was diluted with 9 μl water. Then 5 μlofa (The codon-optimized genesequencewas provided in 5U/μl solution of lyticase was added and incubated at Additional file 2). All restriction endonucleases were from 30°C for 10 min. The sample was frozen at − 80°C for Fermentas (Pittsburgh, Pennsylvania, USA) or Takara Bio- 10 min. A 50 μl PCR was set up using Taq polymerase, technology (Otsu, Shiga, Japan). The kits used for molecu- 5′ AOX1 primer and 3′ AOX1 primer. A 10 μl aliquot lar cloning were from Omega Bio-tek (Norcross, Georgia, was analyze by agarose gel (1%) electrophoresis. USA) or Takara Biotechnology. The expression vector The above nine transformants were screened for pro- pPICZαA was from Invitrogen. Super GelRed was pur- tein expression by a small-scale protein expression fol- chased from US Everbright Inc.. Antibodies and chemical lowing the manufacturer’s protocol. The supernatants reagents used for Western blot were from Tiangen (China). were precipitated with cold acetone, and the precipitated samples were used for SDS-PAGE (12% polyacrylamide Bacterial strains, plasmids, and media gels) analysis. E. coli DH5α was used for routine DNA transformation Five (pJL36A, pJL36C, pJL36E, pJL36G, and pJL36I) and plasmid isolation. P. pastoris GS115 was utilized for out of nine transformants with the higher cellulase ex- cellulase overexpression. E. coli DH5α was routinely pression level were further screened and optimized (in- grown in Luria-Bertani broth at 37°C with aeration or duced for different time intervals). 0.5% Methanol was on LB supplemented with 1.5% (w/v) agar. 25 μg Zeo- added every 24 h until the optimal induction time is cin/ml was added when required. P. pastoris GS115 was reached. The crude protein samples were analyzed by routinely grown in YPD (Yeast Extract Peptone Dextrose SDS-PAGE as above. Medium) at 30°C with aeration or on YPD supple- mented with 1.5% (w/v) agar. For RsEG overexpression, Protein overexpression P. pastoris was first grown overnight in BMGY (buffered The transformant showing the highest protein expres- complex glycerol medium), then in baffled flasks in sion level was used for large-scale expression (100 ml) in BMMY (buffered complex methanol medium) for a baffled flasks according to the standard protocol. Protein couple of days. YPD, BMGY and BMMY were prepared expression was induced with 0.5% methanol for 4 days. according to the standard protocol. The supernatants were harvested by centrifugation at 5000 g and 4°C for 15 min, and precipitated with 80% DNA manipulations (NH ) SO . The precipitated proteins were redissolved 4 2 4 General molecular biology techniques were carried out in buffer A (50 mM Tris-HCl, pH 8.0, 0.5 M NaCl), and by standard procedures [36]. Restriction and modifica- stored at 4 °C. The enzyme purity was analyzed via SDS- tion enzymes were used following the recommendations PAGE. The protein concentration was determined by of the manufacturers. DNA fragments were purified the Bradford method using bovine serum albumin as a Zhang et al. BMC Biotechnology (2018) 18:35 Page 8 of 9 standard. For Western blot, proteins were transferred under different condition were incubated for 15 min. from the gel onto a PVDF membrane. The membrane Specific activities were determined. All enzymatic assays was blocked with 5% (w/v) skimmed milk in TBST were carried out in triplicate. (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.05% Tween To determine the thermal stability of RsEG ,itwas 20), incubated with the murine monoclonal anti- pre-incubated for varied time intervals (15 min to 2 h) polyhistidine immunoglobulin G (IgG), rinsed three at pH 5.0, and 30°C, 40°C and 50°C respectively, and times with TBST, incubated with the goat anti-mouse samples were chilled on ice for at least 10 min. After- IgG conjugated with alkaline phosphatase, rinsed three wards the residual activities were measured under stand- times with TBST, and detected with BCIP (5-bromo-4- ard conditions (optimal pH, 37°C for 15 min). The chloro-3-indolyl phosphate)/NBT (nitro blue tetrazo- experiments at 30°C and 40°C were done with 0.1 mg/ lium) solution. ml of RsEG , whereas the ones at 50°C were done with 0.3 mg/ml of RsEG . The original activity at pH 5.0 and Enzyme activity assay 37°C was taken as 100%, and the percentage of the re- All enzymatic assays were carried out in triplicate. Cellu- sidual activity at different time points and temperatures lase activity was assayed by measuring the amount of re- against the initial one was calculated. ducing sugars released from CMC (Carboxymethyl Cellulose) using the DNS (3,5-dinitrosalicylic acid) Determination of kinetic parameters method [37]. D-Glucose was used as a standard. The Kinetic parameters were determined under initial rate standard assay mixture (1 mL) consisted of 0.5% CMC conditions using non-linear regression analysis of the (w/v) and appropriately diluted enzyme solution in Michaelis–Menten equation. Cellulolytic activity was 50 mM B & R (Britton and Robinson) buffer (pH 5.5), measured at 37°C using CMC as substrate at concentra- and enzymatic reactions were performed at 37°C for tions ranging from 0.2 to 2% (w/v) in a 50 mM B & R 15 min. Reactions were stopped by adding 1.5 ml DNS buffer (pH 5.0). The release of reducing sugars was reagent, followed by boiling for 5 min, then cooled down quantified as above after being incubated for 5 min. All by running tap water. Finally, 2.5 ml of deionized water assays were done in triplicate. was added, and the absorbance at 540 nm was measured. One unit (U) of cellulase activity toward CMC was de- Effects of divalent metal ions on enzyme activity fined as the amount of protein required to release 1 The stimulatory or inhibitory effects of divalent metal μmol of reducing sugar per min under standard assay ions (1 mM) on the catalytic activity of RsEG were de- conditions, and specific activity was defined as units termined by adding 1 mM of various divalent metals − 1 mg protein. (Pb(CH COO) , NiSO , MnSO , CuSO , BaCl , ZnSO , 3 2 4 4 4 2 4 CoCl , CaCl , MgCl and FeSO ) to the standard enzyme 2 2 2 4 Determination of optimal pH and pH stability assay system as above. Since phosphate in B & R buffer The optimal pH of RsEG was evaluated in 50 mM B & might impact the assay, 100 mM sodium acetate (pH 5. R buffer at 37 °C and pH between 3.0 and 11.0 using 0. 0) was used for these assays instead. The system without 5% CMC as the substrates, and all enzymatic reactions supplying divalent metal ions was used as the control. under different condition were incubated for 15 min. The activity of the control at pH 5.0 and 37°C was taken Specific activities were determined. All enzymatic assays as 100%, and the percentage of the activity in the pres- were done in triplicate. ence of different divalent metal ions against the control The pH stability assay was estimated by first pre- was calculated. All enzymatic assays were done in incubating RsEG in 50 mM B & R buffer at different triplicate. pH values (pH 3.0 to 11.0) at 4 °C for 1, 5, 24 and 120 h respectively. The residual activities were then measured Additional files immediately under standard conditions (optimal pH, 37° C for 15 min). The initial activity at optimal pH (5.0) Additional file 1: Sequence alignment of biochemically characterized was taken as 100%, and the percentage of the residual insect-origin GHF9 endocellulases. (DOCX 535 kb) activity at different time points and pH values against Additional file 2: Codon-optimized gene sequence of RsEG from the original one at optimal pH (5.0) was calculated. Reticulitermes speratus with three mutations. (DOCX 14 kb) Additional file 3: SDS-PAGE analysis of overexpressed pJL36A, pJl36C, Determination of optimal temperature and thermal pJL36E, pJL36G and pJL36I after induced for 72 h. (DOCX 577 kb) stability Additional file 4: SDS-PAGE analysis of overexpressed pJL36A, pJl36C and pJL36E over different time intervals. (DOCX 1251 kb) The optimal temperature was determined pH 5.0 or 6.0 Additional file 5: Western blot analysis of overexpressed pJL36C induced (50 mM B & R buffer) between 20 and 65°C using CMC for 72 h. (DOCX 249 kb) (0.5%) as the substrate, and all enzymatic reactions Zhang et al. BMC Biotechnology (2018) 18:35 Page 9 of 9 Abbreviations 16. Woon JS, King PJH, Mackeen MM, Mahadi NM, Wan Seman WMK, BGLs: β-glucosidases; CBHs: Cellobiohydrolases; CMC: Carboxymethyl Cellulose; Broughton WJ, Abdul Murad AM, Abu Bakar FD. Cloning, production and EGLs: Endoglucanases; GHF: Glycoside hydrolase families characterization of a glycoside hydrolase family 7 enzyme from the gut microbiota of the termite Coptotermes curvignathus. Mol Biotechnol. 2017; 59:271–83. Acknowledgements 17. Warnecke F, Luginbuhl P, Ivanova N, Ghassemian M, Richardson TH, Stege We are grateful to the Hi-Tech Research and Development Program of China JT, et al. Metagenomic and functional analysis of hindgut microbiota of a (Grant No. 2014AA093511) and Shandong Province Taishan Scholar Blue wood-feeding higher termite. Nature. 2007;450:560–5. Team Support Project (No. 260 in 2015) for the financial support. 18. Burnum KE, Callister SJ, Nicora CD, Purvine SO, Hugenholtz P, Warnecke F, Scheffrahn RH, Smith RD, Lipton MS. Proteome insights into the symbiotic Availability of data and materials relationship between a captive colony of Nasutitermes corniger and its The datasets supporting the conclusions of this article are included within hindgut microbiome. ISME J. 2011;5:161–4. the article and its additional files. 19. Tokuda G, Lo N, Watanabe H, Slaytor M, Matsumoto T, Noda H. Metazoan cellulase genes from termites: intron/exon structures and sites of Authors’ contributions expression. Biochim Biophys Acta. 1999;1447:146–59. JJL, XY and YD designed the experiments. PZ performed the experiments, 20. Kim N, Choo YM, Lee KS, Hong SJ, Seol KY, Je YH, Sohn HD, Jin BR. including gene cloning, overexpression, characterization, and enzymatic Molecular cloning and characterization of a glycosyl hydrolase family 9 assays. JJL drafted the manuscript. All authors read and approved the final cellulase distributed throughout the digestive tract of the cricket Teleogryllus manuscript. emma. Comp Biochem Physiol B Biochem Mol Biol. 2008;150:368–76. 21. Zhang D, Lax AR, Raina AK, Bland JM. Differential cellulolytic activity of Ethics approval and consent to participate native-form and C-terminal tagged-form cellulase derived from Coptotermes Not applicable. formosanus and expressed in E. coli. Insect Biochem Mol Biol. 2009;39:516–22. 22. Zhang DH, Lax AR, Bland JM, Allen AB. Characterization of a new Competing interests endogenous endo-β-1,4-glucanase of Formosan subterranean termite The authors declare that they have no competing interests. (Coptotermes formosanus). Insect Biochem Mol Biol. 2011;41:211–8. 23. Zhou X, Kovaleva ES, Wu-Scharf D, Campbell JH, Buchman GW, Boucias DG, Scharf ME. Production and characterization of a recombinant β-1,4- Publisher’sNote endoglucanase (glycohydrolase family 9) from the termite Reticulitermes Springer Nature remains neutral with regard to jurisdictional claims in flavipes. Arch Insect Biochem Physiol. 2010;74:147–62. published maps and institutional affiliations. 24. Willis JD, Oppert B, Oppert C, Klingeman WE, Jurat-Fuentes JL. Identification, cloning, and expression of a GHF9 cellulase from Tribolium castaneum Received: 6 December 2017 Accepted: 2 April 2018 (Coleoptera: Tenebrionidae). J Insect Physiol. 2011;57:300–6. 25. Shirley D, Oppert C, Reynolds TB, Miracle B, Oppert B, Klingeman WE, Jurat- Fuentes JL. Expression of an endoglucanase from Tribolium castaneum References (TcEG1) in Saccharomyces cerevisiae. Insect Sci. 2014;21:609–18. 1. Menon V, Rao M. 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Heterologous expression and biochemical characterization of a GHF9 endoglucanase from the termite Reticulitermes speratus in Pichia pastoris

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Abstract

Background: Cellulases are of great significance for full utilization of lignocellulosic biomass. Termites have an efficient ability to degrade cellulose. Heterologous production of the termite-origin cellulases is the first step to realize their industrial applications. The use of P. pastoris for the expression of recombinant proteins has become popular. The endoglucanase from Reticulitermes speratus (RsEG), belonging to glycoside hydrolase family 9 (GHF9), has not been produced in P. pastoris yet. Results: A mutant RsEG (G91A/Y97W/K429A) was successfully overexpressed in P. pastoris. RsEG , with optimum m m pH 5.0, was active over the pH range of 4.0 to 9.0, and exhibited superior pH stability over between pH 4.0 and pH 11.0. It displayed the highest activity and good stability at 40 °C, but lost activity quickly at 50 °C. The apparent kinetic parameters of RsEG against Carboxymethyl Cellulose (CMC) were determined, with K and V of 7.6 mg/ m m max 2+ 2+ 2+ ml and 5.4 μmol/min� mg respectively. Co ,Mn and Fe enhanced the activity of RsEG by 32.0, 19.5 and 11.2% 2+ 2+ respectively, while Pb and Cu decreased its activity by 19.6 and 12.7% separately. Conclusions: RsEG could be overexpressed in P. pastoris. It was stable between pH 4.0 and pH 11.0, and exhibited higher stability at temperatures ≤ 40 °C. This endoglucanase may have potential to be used in the field of laundry, textile and lignocellulose-based biofuels and chemicals. Keywords: Reticulitermes speratus, GHF9 endoglucanse, Heterologous expression, Pichia pastoris, Enzymology Background degradation of cellulose is mainly performed by cellu- Lignocellulosic biomass obtained as agricultural and in- lases produced by microorganisms. To breakdown cellu- dustrial byproducts is an abundant, inexpensive and re- lose efficiently, three classes of cellulases are needed to newable source, and is a desirable feedstock for the work synergistically: endoglucanases (EGLs, EC 3.2.1.4), sustainable production of liquid fuels and chemicals cellobiohydrolases (CBHs, EC 3.2.1.91) and β-glucosidases through the biorefinery processes [1, 2]. Lignocellulose (BGLs, EC 3.2.1.21) [3, 4]. EGLs hydrolyze intramolecular is mainly composed of cellulose, hemicellulose and lig- β-1,4-glucosidic linkages in cellulose randomly, whereas nin, among which cellulose is the major polysaccharide. CBHs cleave cellulose from the reducing and non- The turnover of cellulose plays an important role in glo- reducing ends in a progressive process. BGLs degrade cel- bal carbon cycle for all living organisms. In nature, the lobiose into glucose. According to the CAZy (Carbohy- drate-Active enZYmes) database, where glycosidases are classified according to similarities in the protein sequence * Correspondence: lemomlyty@sohu.com; ygdu@ipe.ac.cn; jjli@ipe.ac.cn and three-dimensional structure, cellulases belong to Sichuan Normal University, College of Life Science, Chengdu 610101, China National Key Laboratory of Biochemical Engineering, National Engineering glycoside hydrolase families (GHF) 5 to 10, 12, 26, 44, 45, Research Center for Biotechnology (Beijing), Key Laboratory of 48, 51, 61 and 74, etc. [5]. Biopharmaceutical Production & Formulation Engineering, PLA, Institute of Process Engineering, Chinese Academy of Sciences, No. 1 North 2nd Street, Beijing 100190, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. BMC Biotechnology (2018) 18:35 Page 2 of 9 Termites (Isoptera or Termitoidae) are the main de- while symbiont cellulases work only in the hindgut [7, 8, graders in tropical and subtropical regions. They have a 15]. The roles of the endogenous cellulases played in the profoundly efficient ability to degrade cellulose [6–8], gut system are postulated to be as important as those and can digest 74 to 99% of the cellulose present in the their symbionts produced (mostly GHF5, GHF7 and plant material they ingest [9]. Thus termite guts are GHF45) in the hindgut for converting cellulose termites regarded as ‘the world smallest bioreactor’ [10, 11]. Ter- ingest [7, 8, 12]. mites are classified into higher- and lower ones based on Heterologous expression of the insect-origin cellulase the presence or absence of flagellated protistan symbi- genes is the first step to realize their industrial applica- onts in their hindguts [7, 8]. Many studies have been tions. Some insect GHF9 endoglucanases have been heter- performed in lower termites. This group of termites con- ologously overexpressed and biochemically characterized. tains a dual cellulose-degradation mechanism: endogen- For instance, RsEG was successfully overexpressed in E. ous cellulases and symbiotic cellulases degrade cellulose coli by using a directed evolution approach, and the ob- cooperatively [7, 8, 12, 13]. All endogenous EGLs exclu- tained mutant A18 was not only efficiently expressed in E. sively belong to the glycoside hydrolase family (GHF) 9 coli, but also showed a 20-fold higher activity than native [7–9, 13–15]. Cellulases of flagellate origin have also RsEG [28, 29]. Later, active RsEG and NtEG were also suc- been identified as members of GHF5, GHF7 and GHF45 cessfully obtained in Aspergillus oryzae [30]. Recombinant from hindgut flagellates of Coptotermes formosanus, C. CfEG3a, Cell-1, CfEG5a, CgEG1 and MbEG1 were also lacteus, Mastotermes darwiniensis and Reticulitermes produced in E. coli, and could hydrolyze cellulose [21–23, speratus [7]. CBHs are only found in the hindgut of 26, 27]. Moreover, active TeEG-I (baculovirus-infected in- lower termites [7, 16], whereas both EGLs and BGLs are sect Sf9 cells) [20], Cell-1 (baculovirus-infected insect Sf9 found in the midgut and hindgut of lower termites [11]. cells) [23], TcEG1 (Drosophila S2 cells and S. cerevisiae) In comparison, higher termites, which do not have fla- [24, 25], and MbEG1 (P. pastoris)were successfullyover- gellates in their hindguts, account for over 75% of ter- expressed in the eukaryotic expression systems [27]. So mite species. The cellulolytic systems of higher termites far, only the kinetic parameters of several insect-origin are different from those of lower ones. Studies have GHF9 endoglucanases were determined, including TeEG- demonstrated that the majority of cellulase activity of I, CfEG3a, RsEG, NtEG, Cell-1, CfEG5 and CgEG1 [20– higher termites takes place in the midgut, suggesting 23, 26, 30]. Eight biochemically characterized insect GHF9 that they mainly depend on endogenous cellulases for endocellulases including RsEG, NtEG, CfEG3a, CfEG5a, cellulose degradation [6, 7, 15]. Metagenomic analysis TcEG1, MbEG1, Cell-1 and TeEG-I were aligned, and the revealed a diverse set of genes related to glycoside hy- sequence identity between them is from 61.6 to 63.2% drolases in the hindgut of a higher termite Nasutitermes (Additional file 1). sp., implying that hindgut microbes also play an import- Reticulitermes speratus is one of the most-extensively ant role for cellulose degradation [17]. Proteome analysis investigated termites in terms of its cellulolytic systems of the bacterial community in a higher termite Nasuti- [7, 8, 14]. Glycoside hydrolases from Reticulitermes sper- termes corniger indicated that bacterial enzymes play atus, belonging to GHF3, GHF7, GHF9 and GHF45, more significant roles in metabolism than in activities were heterologously overexpressed in E. coli, S. crevisiae related to cellulose degradation [18]. and A. oryzae respectively [31, 32]. However, no glycosi- Since identification of an endogenous cellulase gene dases from Reticulitermes speratus have been produced (RsEG)in Reticulitermes speratus by Watanabe et al. in P. pastoris. So far, only one GHF9 endo-glucanase [14], encoding an EGL in GHF9, a lot of insect-origin from termite Macrotermes barneyi was successfully GHF9 cellulase genes have been cloned and/or analyzed, expressed in P. pastoris. Methylotrophic yeast P. pastoris such as NtEG from the higher termite Nasutitermes can strongly over-expresses foreign proteins and serve as takasagoensis [19], NwEG from Nasutitermes walker an expression system for insect proteins [33, 34]. There- [19], TeEG-I from the cricket Teleogryllus emma [20], fore, in this study, an RsEG mutant (G91A/Y97W/ CfEG3a and CfEG5 from Formosan subterranean termite K429A) named as RsEG was heterologously overexpro- (Coptotermes formosanus)[21, 22], Cell-1 from Reticuli- duced in P. pastoris, and recombined RsEGm was fully termes flavipes [23], TcEG1 from red flour beetle Tribo- characterized, including optimal pH and temperature, lium castaneum [24, 25], CgEG1 from the Brazaian pH and thermal stability, kinetic parameters and impact termite Coptotermes gestroi [26], MbEG1 from the of divalent metal ions on the enzymatic activity. fungus-growing higher termite Macrotermes barneyi [27], etc.. These cellulase-coding genes are predomin- Results antly expressed in salivary glands/midgut/foregut [7, 8, Overexpression of RsEG in P. pastoris 15, 23]. Salivary gland−/midgut-secreted endogenous The codon optimized gene RsEG encoding endogluca- cellulases may work in both the midgut and the hindgut nase from Reticulitermes speratus (GenBank: AB008778. Zhang et al. BMC Biotechnology (2018) 18:35 Page 3 of 9 2) with three mutations (G91A/Y97W/K429A) was syn- residual activities at pH 9.0 and pH 10.0 respectively, and thesized [29] (Additional file 2), and cloned into the ex- exhibited very low activity (< 15.0% residual activities) at pression vector pPICZαA at the restriction sites of EcoRI pH 3.0 and pH 11.0. Therefore, RsEG was active over and XbaI. The obtained construct pJL36 was confirmed the pH range of 4.0 to 9.0. Our result is slightly different by DNA sequencing. Linearized construct by BstXI was from the published one for recombinant WT RsEG pro- transformed into P. pastoris GS115 by electroporation. duced in A. oryzae: it had similar optimal pH (pH 5.5) to Nine transformants were randomly picked, grown and RsEG , but exhibited superior activity within a narrow used for PCR. The agarose gel electrophoresis results of pH range (pH 5.0 - pH 7.5) [30]. the PCR products of nine transformants, corresponding The pH stability of RsEG was investigated after being to the size (~1800 bp) of RsEG plus α-factor signal se- pre-incubated for a fixed time over the pH range of 3.0 quence, c-myc epitope and His tag, confirmed that the to 11.0 (Fig. 3). Notably, RsEG was stable over the pH 6 m RsEG gene was successfully inserted into genome P. pas- range of 4.0 to 11.0, retaining more than 74.0% of ori- toris GS115 (Fig. 1). ginal activity after 120 h. It was unstable at pH 3.0, and Five (pJL36A, pJL36C, pJL36E, pJL36G and pJL36I) out only kept 32.5% of initial activity after 120 h. RsEG ex- of nine transformants were used to screen the one with hibited good pH stability over a wide pH range (pH 4.0 - the highest protein expression level. As shown in pH 11.0). The results are very similar to the reported Additional file 3, pJL36A, pJL36C and pJL36E showed ones for recombinant WT RsEG in A. oryzae, in which higher protein production level (Additional file 1:Figure it retained over 80% initial activity after 20 h of pre- S1). According to the amino acid sequence of pJL36 and incubation between pH 5.0 and 10.0, and lost activity the C-terminal peptide containing c-myc epitope and His sharply at pH 3.0 [30]. tag, the predicted molecular weight of pJL36 is around 48. 7 kDa, which was much lower than the apparent molecu- Determination of optimal temperature and thermal lar weight on SDS-PAGE (Additional file 3). The differ- stability of RsEG ence between the predicted molecular weight and the The optimal temperature of RsEG was determined apparent one for pJL36 was possibly due to the fact that (Fig. 4). It showed the highest activity at 40 °C, and highly glycosylated proteins are usually obtained when kept > 72.8% of residual activity between 20 and 45 °C. they are overexpressed in P. pastoris [32]. After pJL36A, However, it lost activity rapidly when temperature rose pJl36C and pJL36E were further induced with 0.5% up to 50°C, retaining only 27.7% of original activity. It methanol for different time intervals, it was found maintained only 18.4% of residual activity at 65°C. Our pJL36C produced higher protein yield and used for sub- result is very similar to the published one for recombin- sequent large-scale overexpression (Additional file 4). ant WT RsEG in A. oryzae, where it had optimal activ- Overexpression of pJL36C was also confirmed by west- ity at 45°C, and lost activity sharply at temperatures ern blotting (Additional file 5) and CMC activity. above 50°C [30]. The thermal stability of RsEG was investigated after Determination of pH optima and pH stability of RsEG being pre-incubated for a fixed time at pH 5.0, and at Britton-Robinson (B & R) buffer is a “universal” buffer 30, 40 and 50 °C respectively (Fig. 5). RsEG was stable used for the range of pH 3.0 to pH 11.0, so it was chosen at 30°C, and lost only 5.5% of original activity after for determining optimal pH and pH stability of RsEG . 120 h of pre-incubation. It kept 66.0% of initial activity As shown in Fig. 2, RsEG showed the highest activity at 40°C after 120 h. It was completely inactivated at 50 ° at pH 5.0, and retained original activities above 77.6% be- C after 120 h, and retained only 15.1% of original activity tween pH 4.0 and pH 8.0. It maintained 66.3 and 48.0% at 50°C after 1 h. Therefore, RsEG was a thermolabile Fig. 1 Agarose gel for PCR products of nine randomly picked transformants. Lane 1: DNA ladder; Lanes 2-10: PCR products of 9 randomly picked transformants (pJL36A - pJL36I) Zhang et al. BMC Biotechnology (2018) 18:35 Page 4 of 9 Fig. 2 Effects of pH on RsEG activity. Values are expressed as the means of three replicates ± standard deviation. The activity of the enzyme at pH 5.0 and 37 °C (6.7 U/mg) was defined as 100% endocellulase, and was fairly stable below 30°C. The re- Effects of divalent metal ions on enzyme activity sults are very similar to the reported ones for recombin- The effects of divalent metal ions on RsEG activity were 2+ 2+ 2+ ant WT RsEG in A. oryzae, where it retained over 80% examined (Fig. 7). Co ,Mn and Fe upregulated the of maximum activity after 30 min-incubation at temper- cellulolytic activity of RsEG by 32.0, 19.5 and 11.2% 2+ 2+ atures as low as 45°C and was not stable at temperatures respectively, while Pb and Cu decreased the activity of above 50°C [30]. RsEG by 19.6 and 12.7% separately. Other divalent metal ions didn’t show obvious influence on the catalytic activity Determination of kinetic parameters of RsEG of RsEG . The kinetic parameters of recombinant RsEG against CMC were determined by substrate hydrolysis from 0.2 to Discussion 2% (w/v) for 5 min. The deduced kinetic values were ap- In the present study, a GHF9 endoglucanase RsEG mu- parent parameters since saturation was not achieved even tant (RsEG ) from Reticulitermes speratus was overex- when high CMC concentrations were used (Fig. 6). The pressed in P. pastoris and characterized. BLAST (Basic apparent K and V values of RsEG towards CMC were Local Alignment Search Tool) search of RsEG resulted m max 7.6 mg/ml and 5.4 μmol/min•mg respectively. in significant matches of GHF9 cellulases originating Fig. 3 pH stability of RsEG . The pH stability assay was investigated by first pre-incubating RsEG in 50 mM B & R buffer at different pH values m m (pH 4.0 to 11.0) at 4°C for 1 h, 5 h, 24 h, and 120 h respectively. The residual activities were then measured immediately under standard conditions (optimal pH, 37°C for 15 min). The initial activity at optimal pH (5.0) and 37°C (6.7 U/mg) was taken as 100%, and the percentage of the residual activity at different time points and pH values against the original one at optimal pH (5.0) was calculated. Values are expressed as the means of three replicates ± standard deviation Zhang et al. BMC Biotechnology (2018) 18:35 Page 5 of 9 Fig. 4 Effects of temperature on RsEG activity. Values are expressed as the means of three replicates ± standard deviation. The original activity at pH 5.0 and 40 °C was taken as 100% (7.9 U/mg) from insects. Among top 100 Blast Hits, only nine endo- were even expressed in the complicated baculovirus ex- cellulaseas were heterologously overexpressed and partly pression system [23, 24]. characterized. As far as we know, this is the first report In comparison with other insect GHF9 endo- on heterologous expression of active RsEG in P. pastoris. cellulaseas, RsEG exhibited higher specific activity than Until now, only one insect endo-cellulase belonging to Cell-1 (~1 U/mg) and comparable activity to crude GHF9, MbEG, was heterologously overexpressed in P. CfEG3a (14-19 U/mg) [21, 23], and much lower specific pastoris [27]. Though active WT RsEG was not pro- activity than other characterized insect GHF9 endoglu- duced in E. coli and S. cerevisiae, an active mutant A18 canases such as recombinant RsEG in A. oryzae (112 U/ was successfully obtained through DNA shuffling of four mg), CfEG5 (325 U/mg), NtEG (105 U/mg), MbEG1 orthologous parental cDNAs in E. coli [28]. Active re- (223.9 U/mg) and TeEG1 (948.1 U/mg) [20, 22, 27, 30]. combinant RsEG was also successfully overexpressed in The lower activity of RsEG recombined in P. pastoris A. oryzae [30]. So far, most insect GHF9 endoglucanases could be presumably due to the reason that RsEG pro- were produced in E. coli [21–23, 26, 27], and several duced in P. pastoris was not correctly folded and/or glycosylated. It appears that most termite GHF9 endoglucanases had optimal pH value around 5.0-6.0, including CfEG3a from Coptotermes formosanus (pH 5.0) [21], MbEG1 from Macrotermes barneyi (pH 5.5) [27], CfEG5 from Coptotermes formosanus (pH 5.6) [22], NtEG from Fig. 5 Thermal stability of RsEG .RsEG was pre-incubated for varied m m times (15 min to 2 h) at pH 5.0, and 30°C, 40°C and 50°C respectively, and samples were chilled on ice for at least 10 min. Afterwards the residual activities were measured under standard conditions (optimal pH, 37°C for 15 min). The original activity at pH 5.0 and 37°C (6.7 U/ mg) was taken as 100%, and the percentage of the residual activity at different time points and temperatures against the initial one was calculated. Values are expressed as the means of three replicates ± Fig. 6 Effects of CMC concentration on RsEG activity. Values are standard deviation expressed as the means of three replicates ± standard deviation Zhang et al. BMC Biotechnology (2018) 18:35 Page 6 of 9 Fig. 7 Effects of divalent metal ions on RsEG activity. The effects of metal ions on the catalytic activity of RsEG were determined by adding m m 1 mM of various divalent metals to the standard enzyme assay system (pH 5.0, 37°C for 15 min). The activity at pH 5.0 and 37°C (6.7 U/mg) in the absence of divalent metal ions (control) was taken as 100%, and the percentage of the activity in the presence of different divalent metal ions against the control was calculated. Values are expressed as the means of three replicates ± standard deviation Nasutitermes takasagoensis (pH 6.0) [30], and CgEG1 incubation at 50°C, and dramatically lost activity at tem- from Coptotermes gestroi (pH 6.0) [26]. The optimum peratures above 55°C [27]. The activity TeEG-I was stable pH of TeEG-I from the cricket Teleogryllus emma with up to 45°C during 10 min of incubation, but was lost after 61% sequence identity to RsEG, was determined to be 5. 10 min-incubation at 75°C [20]. The t values of CgEG1 1/2 0[20]. By contrast, recombinant TcEG1 from red flour at 45, 50, 60 and 70 °C were 46.21, 8.2, 1.48 and 0.35 min, beetle Tribolium castaneum showed the maximum ac- respectively [26]. NtEG retained 60% of maximum activity tivity at pH 12.0. Only one neutral insect-origin GHF9 after 30 min of pre-incubation at temperatures as low as endocellulase – Cell-11, from the termite Reticulitermes 60°C, and lost activity sharply above 60°C [30]. Thus, it flavipes, was identified and characterized, with optimal seems that NtEG and MbEG1 were thermally stable than pH between 6.5 and 7.5 [23]. Therefore, most biochem- RsEG, whereas it displayed comparable thermal stability ically characterized insect GHF9 endoglucanases were to TeEG-I and CgEG1 [20, 26, 27, 30]. classified as acidic cellulases. Comparison of the charac- In the case of the kinetic parameters of insect GHF9 terized insect GHF9 endoglucanases revealed that most endocellulases, the apparent K value of RsEG is com- m m of them were active over a wide pH range, including parable to that of Cell-1 expressed by BEVS (9.9 mg/ml) RsEG (pH 4.0 - pH 9.0), CfEG5 (pH 3.6 - pH 9.6), and in E. coli (14.7 mg/ml) [23], and is much higher than TeEG-I (pH 3.0 - pH 8.5) and Cell-1 (pH 4.0 - pH 9.0) those of WT RsEG expressed in A. oryzae (2.0 mg/ml), [20, 22, 23]. In contrast, MbEG1 (pH 4.5 - pH 6.5), CfEG3a (2.2 mg/ml), NtEG (4.7 mg/ml), and CfEG5 (5. TcEG1 (pH 8.0 - pH 12.0), and RsEG produced in A. 6 mg/ml) [21, 22, 30]. Therefore, it showed lower bind- oryzae showed higher activity over a narrow pH range ing affinity towards CMC. Although its V value was max (pH 5.0 - pH 7.5) [27, 30]. Moreover, RsEG exhibited much higher than those of Cell-1 produced by BEVS (1. higher thermal stability than recombinant WT NtEG in 06 μmol/min•mg) and in E. coli (0.84 μmol/min•mg) A. oryzae and MbEG, which retained over 75% of max- [23], it was greatly lower than those of WT RsEG imal activity after 30 min of incubation at pH 3.5 to expressed in A. oryzae (1429 U/mg) [30], CfEG3a pH 8.0 and was stable after 20 h of pre-incubation be- (590 U/mg) [21], NtEG (1667 U/mg) [30], and CfEG5 tween pH 5.5 and pH 9.0 respectively [27, 30]. (548 U/mg) [22]. Just like RsEG , TeEG-I, CfEG3a, CfEG5, MbEG1 and A lot of studies towards impact of divalent metal ions recombinant TcEG1 had highest enzymatic activity at on endo-cellulases were done [35]. However, until now, around 40°C [20–22, 25, 27]. The optimal temperatures there was only one report on insect-origin GHF9 endo- 2+ of CgEG1, and Cell1 were 50°C and in the range of 50- glucanases that Ca slightly enhanced CMC activity of 60°C respectively [23, 26]. Recombinant NtEG in A. ory- Cell-1, and stabilized its activity over time [23]. In our 2+ zae had optimal activity at 65°C [30]. Until now, only study, Ca didn’t show obvious impact on RsEG . the thermal stabilities of several insect GHF9 endogluca- Therefore, it seems that same metal ions had different nases were investigated. For example, MbEG1 retained influence on GHF9 endoglucanases from different 60% of its maximum activity after 30 min of pre- species of insects. Zhang et al. BMC Biotechnology (2018) 18:35 Page 7 of 9 Conclusions from agarose gels using the DNA gel extraction kit. Plas- In summary, a GHF9 endoglucanase RsEG mutant mid DNA was isolated using the plasmid miniprep kit. (RsEG ) from Reticulitermes speratus was successfully The plasmid for the synthesized RsEG gene with the m m recombined in P. pastoris and characterized in detail. restriction sites of EcoRI and XbaIat5′ and 3′-terminal Recombinant RsEG showed the highest activity at respectively, which was cloned into PUC57, was digested pH 5.0 and 40 °C, and was very stable between pH 4.0 with restriction enzymes EcoRI and XbaI, and re-cloned and pH 11.0. It exhibited higher stability at tempera- into the vector pPICZαA digested with EcoRI and XbaI, tures ≤ 40°C and was unstable above 45°C. The appar- respectively. The final construct was confirmed by DNA ent K and V values of RsEG against CMC were sequencing, and named as pJL36. m max m 2+ 7.6 mg/ml and 5.4 μmol/min•mg respectively. Co , 2+ 2+ Mn and Fe showed some stimulatory effects on Screening of recombinant colonies by direct PCR and 2+ 2+ RsEG ,while RsEG was inhibited by Pb and Cu . expression m m Therefore, RsEG exhibited good pH and thermal stability The construct pJL36 was linearized with BstXI and to an extent, and activities of insect endoglucanases may transformed into P. pastoris GS115 by electroporation be enhanced by some metal ions. following the manufacturer’s recommendation. Zeocin- resistant P. pastoris clones were grown on YPDS plates Methods containing 100 μg/ml Zeocin. 10-20 colonies were Materials picked and streaked for single colonies on fresh YPD or Chemicals were from Sigma, Merck or Ameresco. Oligonu- YPDS plates containing 100 μg/ml of Zeocin. cleotides and the codon-optimized RsEG gene encoding Nine transformants were randomly selected and grown an endo-β-1,4-glucanase from the termite Reticulitermes overnight in YPD. 10 μlof a Pichia pastoris culture was speratus with three mutations (G91A/Y97W/K429A) were placed into a 1.5 ml microcentrifuge tube, and 1 μlof synthesized by Shanghai Sangon Biotech Co. Ltd. (China) the culture was diluted with 9 μl water. Then 5 μlofa (The codon-optimized genesequencewas provided in 5U/μl solution of lyticase was added and incubated at Additional file 2). All restriction endonucleases were from 30°C for 10 min. The sample was frozen at − 80°C for Fermentas (Pittsburgh, Pennsylvania, USA) or Takara Bio- 10 min. A 50 μl PCR was set up using Taq polymerase, technology (Otsu, Shiga, Japan). The kits used for molecu- 5′ AOX1 primer and 3′ AOX1 primer. A 10 μl aliquot lar cloning were from Omega Bio-tek (Norcross, Georgia, was analyze by agarose gel (1%) electrophoresis. USA) or Takara Biotechnology. The expression vector The above nine transformants were screened for pro- pPICZαA was from Invitrogen. Super GelRed was pur- tein expression by a small-scale protein expression fol- chased from US Everbright Inc.. Antibodies and chemical lowing the manufacturer’s protocol. The supernatants reagents used for Western blot were from Tiangen (China). were precipitated with cold acetone, and the precipitated samples were used for SDS-PAGE (12% polyacrylamide Bacterial strains, plasmids, and media gels) analysis. E. coli DH5α was used for routine DNA transformation Five (pJL36A, pJL36C, pJL36E, pJL36G, and pJL36I) and plasmid isolation. P. pastoris GS115 was utilized for out of nine transformants with the higher cellulase ex- cellulase overexpression. E. coli DH5α was routinely pression level were further screened and optimized (in- grown in Luria-Bertani broth at 37°C with aeration or duced for different time intervals). 0.5% Methanol was on LB supplemented with 1.5% (w/v) agar. 25 μg Zeo- added every 24 h until the optimal induction time is cin/ml was added when required. P. pastoris GS115 was reached. The crude protein samples were analyzed by routinely grown in YPD (Yeast Extract Peptone Dextrose SDS-PAGE as above. Medium) at 30°C with aeration or on YPD supple- mented with 1.5% (w/v) agar. For RsEG overexpression, Protein overexpression P. pastoris was first grown overnight in BMGY (buffered The transformant showing the highest protein expres- complex glycerol medium), then in baffled flasks in sion level was used for large-scale expression (100 ml) in BMMY (buffered complex methanol medium) for a baffled flasks according to the standard protocol. Protein couple of days. YPD, BMGY and BMMY were prepared expression was induced with 0.5% methanol for 4 days. according to the standard protocol. The supernatants were harvested by centrifugation at 5000 g and 4°C for 15 min, and precipitated with 80% DNA manipulations (NH ) SO . The precipitated proteins were redissolved 4 2 4 General molecular biology techniques were carried out in buffer A (50 mM Tris-HCl, pH 8.0, 0.5 M NaCl), and by standard procedures [36]. Restriction and modifica- stored at 4 °C. The enzyme purity was analyzed via SDS- tion enzymes were used following the recommendations PAGE. The protein concentration was determined by of the manufacturers. DNA fragments were purified the Bradford method using bovine serum albumin as a Zhang et al. BMC Biotechnology (2018) 18:35 Page 8 of 9 standard. For Western blot, proteins were transferred under different condition were incubated for 15 min. from the gel onto a PVDF membrane. The membrane Specific activities were determined. All enzymatic assays was blocked with 5% (w/v) skimmed milk in TBST were carried out in triplicate. (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.05% Tween To determine the thermal stability of RsEG ,itwas 20), incubated with the murine monoclonal anti- pre-incubated for varied time intervals (15 min to 2 h) polyhistidine immunoglobulin G (IgG), rinsed three at pH 5.0, and 30°C, 40°C and 50°C respectively, and times with TBST, incubated with the goat anti-mouse samples were chilled on ice for at least 10 min. After- IgG conjugated with alkaline phosphatase, rinsed three wards the residual activities were measured under stand- times with TBST, and detected with BCIP (5-bromo-4- ard conditions (optimal pH, 37°C for 15 min). The chloro-3-indolyl phosphate)/NBT (nitro blue tetrazo- experiments at 30°C and 40°C were done with 0.1 mg/ lium) solution. ml of RsEG , whereas the ones at 50°C were done with 0.3 mg/ml of RsEG . The original activity at pH 5.0 and Enzyme activity assay 37°C was taken as 100%, and the percentage of the re- All enzymatic assays were carried out in triplicate. Cellu- sidual activity at different time points and temperatures lase activity was assayed by measuring the amount of re- against the initial one was calculated. ducing sugars released from CMC (Carboxymethyl Cellulose) using the DNS (3,5-dinitrosalicylic acid) Determination of kinetic parameters method [37]. D-Glucose was used as a standard. The Kinetic parameters were determined under initial rate standard assay mixture (1 mL) consisted of 0.5% CMC conditions using non-linear regression analysis of the (w/v) and appropriately diluted enzyme solution in Michaelis–Menten equation. Cellulolytic activity was 50 mM B & R (Britton and Robinson) buffer (pH 5.5), measured at 37°C using CMC as substrate at concentra- and enzymatic reactions were performed at 37°C for tions ranging from 0.2 to 2% (w/v) in a 50 mM B & R 15 min. Reactions were stopped by adding 1.5 ml DNS buffer (pH 5.0). The release of reducing sugars was reagent, followed by boiling for 5 min, then cooled down quantified as above after being incubated for 5 min. All by running tap water. Finally, 2.5 ml of deionized water assays were done in triplicate. was added, and the absorbance at 540 nm was measured. One unit (U) of cellulase activity toward CMC was de- Effects of divalent metal ions on enzyme activity fined as the amount of protein required to release 1 The stimulatory or inhibitory effects of divalent metal μmol of reducing sugar per min under standard assay ions (1 mM) on the catalytic activity of RsEG were de- conditions, and specific activity was defined as units termined by adding 1 mM of various divalent metals − 1 mg protein. (Pb(CH COO) , NiSO , MnSO , CuSO , BaCl , ZnSO , 3 2 4 4 4 2 4 CoCl , CaCl , MgCl and FeSO ) to the standard enzyme 2 2 2 4 Determination of optimal pH and pH stability assay system as above. Since phosphate in B & R buffer The optimal pH of RsEG was evaluated in 50 mM B & might impact the assay, 100 mM sodium acetate (pH 5. R buffer at 37 °C and pH between 3.0 and 11.0 using 0. 0) was used for these assays instead. The system without 5% CMC as the substrates, and all enzymatic reactions supplying divalent metal ions was used as the control. under different condition were incubated for 15 min. The activity of the control at pH 5.0 and 37°C was taken Specific activities were determined. All enzymatic assays as 100%, and the percentage of the activity in the pres- were done in triplicate. ence of different divalent metal ions against the control The pH stability assay was estimated by first pre- was calculated. All enzymatic assays were done in incubating RsEG in 50 mM B & R buffer at different triplicate. pH values (pH 3.0 to 11.0) at 4 °C for 1, 5, 24 and 120 h respectively. The residual activities were then measured Additional files immediately under standard conditions (optimal pH, 37° C for 15 min). The initial activity at optimal pH (5.0) Additional file 1: Sequence alignment of biochemically characterized was taken as 100%, and the percentage of the residual insect-origin GHF9 endocellulases. (DOCX 535 kb) activity at different time points and pH values against Additional file 2: Codon-optimized gene sequence of RsEG from the original one at optimal pH (5.0) was calculated. Reticulitermes speratus with three mutations. (DOCX 14 kb) Additional file 3: SDS-PAGE analysis of overexpressed pJL36A, pJl36C, Determination of optimal temperature and thermal pJL36E, pJL36G and pJL36I after induced for 72 h. (DOCX 577 kb) stability Additional file 4: SDS-PAGE analysis of overexpressed pJL36A, pJl36C and pJL36E over different time intervals. (DOCX 1251 kb) The optimal temperature was determined pH 5.0 or 6.0 Additional file 5: Western blot analysis of overexpressed pJL36C induced (50 mM B & R buffer) between 20 and 65°C using CMC for 72 h. (DOCX 249 kb) (0.5%) as the substrate, and all enzymatic reactions Zhang et al. BMC Biotechnology (2018) 18:35 Page 9 of 9 Abbreviations 16. Woon JS, King PJH, Mackeen MM, Mahadi NM, Wan Seman WMK, BGLs: β-glucosidases; CBHs: Cellobiohydrolases; CMC: Carboxymethyl Cellulose; Broughton WJ, Abdul Murad AM, Abu Bakar FD. Cloning, production and EGLs: Endoglucanases; GHF: Glycoside hydrolase families characterization of a glycoside hydrolase family 7 enzyme from the gut microbiota of the termite Coptotermes curvignathus. Mol Biotechnol. 2017; 59:271–83. Acknowledgements 17. Warnecke F, Luginbuhl P, Ivanova N, Ghassemian M, Richardson TH, Stege We are grateful to the Hi-Tech Research and Development Program of China JT, et al. Metagenomic and functional analysis of hindgut microbiota of a (Grant No. 2014AA093511) and Shandong Province Taishan Scholar Blue wood-feeding higher termite. Nature. 2007;450:560–5. Team Support Project (No. 260 in 2015) for the financial support. 18. Burnum KE, Callister SJ, Nicora CD, Purvine SO, Hugenholtz P, Warnecke F, Scheffrahn RH, Smith RD, Lipton MS. Proteome insights into the symbiotic Availability of data and materials relationship between a captive colony of Nasutitermes corniger and its The datasets supporting the conclusions of this article are included within hindgut microbiome. ISME J. 2011;5:161–4. the article and its additional files. 19. Tokuda G, Lo N, Watanabe H, Slaytor M, Matsumoto T, Noda H. Metazoan cellulase genes from termites: intron/exon structures and sites of Authors’ contributions expression. Biochim Biophys Acta. 1999;1447:146–59. JJL, XY and YD designed the experiments. PZ performed the experiments, 20. Kim N, Choo YM, Lee KS, Hong SJ, Seol KY, Je YH, Sohn HD, Jin BR. including gene cloning, overexpression, characterization, and enzymatic Molecular cloning and characterization of a glycosyl hydrolase family 9 assays. JJL drafted the manuscript. All authors read and approved the final cellulase distributed throughout the digestive tract of the cricket Teleogryllus manuscript. emma. Comp Biochem Physiol B Biochem Mol Biol. 2008;150:368–76. 21. Zhang D, Lax AR, Raina AK, Bland JM. Differential cellulolytic activity of Ethics approval and consent to participate native-form and C-terminal tagged-form cellulase derived from Coptotermes Not applicable. formosanus and expressed in E. coli. Insect Biochem Mol Biol. 2009;39:516–22. 22. Zhang DH, Lax AR, Bland JM, Allen AB. Characterization of a new Competing interests endogenous endo-β-1,4-glucanase of Formosan subterranean termite The authors declare that they have no competing interests. (Coptotermes formosanus). 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Published: Jun 1, 2018

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