The gene of malPQ encoding 4-α-glucanotransferase (amylomaltase) is located in the malPQ operon of Escherichia coli K12. e reactions of the enzyme with several types of carbohydrate were carried out under the optimal conditions in an effort to understand the function of MalPQ enzyme in maltodextrin and glycogen metabolism of E. coli.
Vietnam Academy of Agricultural Sciences (VAAS) General Statistic O ce, 2018 Statistical year book of Vietnam 2017 Statistical Publishing House, Hanoi General Statistic O ce, 2017 Statistical year book of Vietnam 2016 Statistical Publishing House, Hanoi Hung P.T., 2016 Overview on green asparagus Department of Sciences and Technology, Ninh uan province Report Jaramillo-Carmona S., Rodriguez-Arcos R., JiménezAraujo A., López S, Gil J., Moreno R., GuillénBejarano R., 2017 Saponin pro le of wild asparagus species J Food Sci., 82: 638-646 Lu G., Jian W., Zhang J., Zhou Y., Cao J., 2008 Suppressive e ect of silicon nutritionon Phomopsis stem blightdevelopment HortScience, 43: 811-817 Maeda T, Kakuta H, Sonoda T, Motoki S, Ueno R, Suzuki T, Oosawa K., 2005 Anti-oxidation capacities of extracts from green, purple, and white asparagus spears related to polyphenol concentration HortScience, 40: 1221-1224 Sonoda T., Uragami A., Kaji K., 1997 Evaluation on Asparagus o cinalis cultivars for resistance to stem blight by using a novel inoculation method HortScience, 32: 1085-1086 Xiang J., Xiang Y., Lin S., Xin D., Liu X ,Weng L., Zhang M., 2014 Anticancer e ects of deproteinized asparagus polysaccharide on hepatocellular carcinoma in vitro and in vivo Tumor Biol., 35: 3517-3524 Zaw M., Naing T.A.A., Matsumoto M., 2017 First report of stem blight of asparagus caused by Phomopsis Asparagi in Myanmar New diseases report 35: 17 Zhang Y., Qu H., Zhao P., Wu L., Zhou J Tang Y Lou S., Chen G., 2018 Transformation of Phomopsis asparagi with green uorescent protein using protoplasts Can J Plant Pathol., 4: 254-260 Date received: 23/11/2018 Date reviewed: 2/12/2018 Reviewer: Dr To i u Ha Date approved for publication: 21/12/2018 ACTION MODES OF MALQ ISOLATED FROM Escherichia coli K12 Tran Phuong Lan*1, Nguyen Minh uy2 Abstract e gene of malQ encoding 4-α-glucanotransferase (amylomaltase) is located in the malPQ operon of Escherichia coli K12 e reactions of the enzyme with several types of carbohydrate were carried out under the optimal conditions in an e ort to understand the function of MalQ enzyme in maltodextrin and glycogen metabolism of E coli e enzyme catalyzed the hydrolysis of the α-1,4 glucosidic linkage of linear maltodextrins, released the reducing-end glucose of dextrins, and it also transferred glycosyl residues onto the non-reducing end of an acceptor via a disproportionation reaction e smallest substrate that MalQ recognized of this reaction mode was maltose Glucose was not the substrate but the great acceptor for this enzyme e enzyme performed intramolecular transglycosylation to produce the cyclic form having degree of polymerization (DP) from DP20 to DP33 with DP24 as the main product in the reaction with amylose substrate ese data may explain the understanding of MalQ mechanism in vivo Keywords: MalQ, 4-α-glucanotransferase (amylomaltase), Escherichia coli, cycloamylose, transglycosylation INTRODUCTION MalQ, known as 4-α-glucanotransferase or amylomaltase, is a member of the α-amylase family and is widely distributed in plants and microorganisms where it is involved in starch metabolism (Sato et al., 2013; Critchley et al., 2001) or maltooligosaccharide metabolism (Boos and Shuman, 1998) MalQ was rst reported as disproportionating enzyme (D-enzyme) in potato tubers by Peat et al (1956) and was reported to occur in a wide range of plant tissues by Lin and Preiss (1988) Early studies focusing on the biochemical properties of D-enzymes illustrated that the multiple D-enzymes have common reaction characteristics MalQ appears to have different physiological functions in microorganisms This enzyme was rst described by Monod and Torriani (1948) as a maltose-inducible enzyme Since then, its gene has been cloned into several bacteria, and homologous genes have been identi ed in several other bacterial genomes According to Kaper et al (2005) the enzyme catalyzes the transfer of the α-1,4-glucan segment from one α-1,4-glucan molecule (donor) to another α-1,4-glucan molecule (acceptor), as expressed in the following equation: Faculty of Agriculture and Natural Resourses, An Giang University College of Agriculture and Applied Biology, Can o University * Corresponding author: Tran Phuong Lan E-mail: tplancntp@gmail.com/ tplan@agu.edu.vn 82 Journal of Vietnam Agricultural Science and Technology - No.1(3)/2018 (α-1,4-glucan)m + (α-1,4-glucan)n (α-1,4-glucan)m-x + (α-1,4-glucan)n + x is is an intermolecular transglycosylation reaction and is o en referred as a disproportionation reaction is enzyme also catalyzes an intramolecular glucan transfer reaction within a single linear glucan molecule to produce cyclic α-1,4-glucan (cycloamylose) as follows: (α-1,4-glucan)n cyclic (α-1,4-glucan)x + (α-1,4-glucan)n- x Recently, several MalQ proteins from various sources have been investigated, such as Arabidopsis (Critchley et al., 2001), Chlamydomonas reinhardtii (Buschiazzo et al., 2004), Potato (Takaha et al., 1996), Escherichia coli ML (Palmer et al., 1976), ermus aquaticus (Przylas et al., 2000), ermus thermophilus (Binnema et al., 1998), Aquifex aeolicus (Bhuiyan et al., 2003), ermotoga martima (Lee et al., 2002), ermococcus litoralis (Jeon et al., 1997), and ermococcus kodakaraensis (Tachibana et al., 2000) e MalQ encoded by malQ gene isolated from E coli K12 has 694 amino acid residues Its molecular protein weight is 72 kDa is enzyme has highest activity at 37°C and pH 6.5 in 50 mM sodium acetate (Tran et al., 2011) is study focused on clari cation of action modes of MalQ encoded by the malQ gene from E coli K12 as a test of E coli matodextrin metabolism in vitro MATERIALS AND METHODS Materials Recombinant E coli carrying malQ gene of E coli mutant K12, kindly provided by Prof W Boos at University of Konstanz, Germany was used to produce MalQ Bovine glycogen, glucose, maltose, maltotriose and maltoheptaose were bought from Sigma Chemical Co (St Louis, USA) Isoamylase isolated from Pseudomanas amyloderamosa was obtained from Hayashibara Biochemical Laboratory (Okayama, Japan) Other reagents used in this study were purchased from Sigma Chemical Co (USA), Merck (Germany), Junsei Chemical Co (Tokyo, Japan), or Showa Chemicals Inc (Tokyo, Japan) Methods reached 0,6 e IPTG was added to the nal concentration of 0.2 mM e culture was then continued to incubate for 6hrs at 25°C and the cells were collected by centrifugation (4,000 ˟ g, 20 min, 4°C) e cell pellet was resuspended in 100 ml of lysis bu er [50 mM tris-HCl (pH 7.5), 300 mM NaCl, 10 mM imidazole] and sonicated over an ice batch (VC-600, Sonics & Materials Inc., Newtown, CT, USA; output 4, ˟ times, 60% duty) e crude cell extract was centrifuged (10,000 ˟ g, 4°C, 15 min) Ten milliliters of the crude enzyme was applied to ml of nickel-nitrilotriacetic acid (Ni-NTA) resin (QIAGEN, USA) packed in a Poly-Prep Chromatography column (BIO-RAD, Hercules, CA, USA) e resin was washed twice with ml of washing bu er [50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 20 mM imidazole] Then ml of elution buffer [50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 250 mM imidazole] was used to allow elution of the target proteins The eluted target protein was concentrated by ultrafiltration (Amicon Co., USA), and dialyzed against 50 mM Tris-HCl bu er (pH 7.5) Determination of protein amount and molecular mass Protein concentration was determined by the Bradford method (Bradford, 1976) e purity and molecular mass of puri ed proteins were analyzed by 10% SDS-PAGE Enzyme reaction Reaction modes of MalQ were carried out by the separated reactions of the enzyme with glucose, maltose, maltotriose, maltoheptaose and amylose in the optimal conditions determined by Tran et al (2011) A er inactivation of the enzyme reaction by boiling, the mixtures were analyzed by thin layer chromatography (TLC) and/or high performance anion exchange chromatography (HPAEC) to understand the enzyme action mechnism in layer chromatography (TLC) analysis TLC was conducted as Tran et al (2014) described with slightly modi cation Isopropyl alcohol - ethyl acetate - water (3 : : 1, v/v/v) or n-butanol - acetic acid - water (5 : : 1, v/v/v) were used as solvent in the TLC method Protein puri cation High performance anion exchange chromatography (HPAEC) analysis In batch puri cation of MalQ, E coli BL21 (DE3) carrying the corresponding gene was cultured in liter of LB broth supplemented with kanamycin 20 ml/ml at 37°C and 200 rpm until the OD600nm e reaction mixtures were boiled for 10 and centrifuged at 12,000 ˟ g for 10 min, and ltered using a membrane lter kit (0.45 mm pore diameter, Gelman Sciences, USA) HPAEC was performed 83 Vietnam Academy of Agricultural Sciences (VAAS) using a Dionex (Sunnyvale, CA, USA) DX-500 system with a pulsed amperometric detector (ED40, Dionex, USA) e system was equipped with a CarboPac PA-100 column (4 ˟ 250 mm, Dionex) and run with a gradient of - 0.6 M sodium acetate in 0.15 M NaOH at a ow rate of 1.0 ml/min (Tran et al., 2015) Time and place of the study Matrix Assisted Laser Desorption Ionization - Time of Flight (MALDI-TOF) mass spectrometry analysis Intermolecular transglycosylation e study was carried out at Seoul National University (South Korea), An Giang University and Can o University (Vietnam) in 2015 RESULTS AND DISCUSSION Molecular mass spectrum of the puri ed enzyme was collected by MALDI-TOF Mass spectrometer [VoyagerTM-DE (Perceptive Biosystem, Framingham, USA)] Sinapinic acid (3,5-dimethoxy-4 hydrony cinnamic acid) is proper matrix for proteins or peptides more than 10 kDa Proper matrix concentration was 10 mg/ml, nal concentration to 0.1 ~ pmol/ml Ten milligram of sinapicnic acid was dissolved in ml of solvents containing 400 ml deionized water, 100 ml 3% TFA and 500 ml acetonitril For the calibration analysis, 1% bovine serum albumin (Sigma) solution was prepared Ten milligram of BSA was dissolved in M of solvent containing 600 ml deionized water, 100 ml 3% TFA and 300 ml acetonitril One microliter of puri ed BBMA (0.1 to 0.01 mg/ml nal concentration) and ml of prepared matrix were mixed and dropped on a sample plate and air dried until crystallization occurred en the sample plate was loaded into the Voyager Biospectrometry Workstation and analyzed the molecular weight of the sample at the proper conditions (Grid voltage: 87, Grid wire voltage: 0.3, Delayed extraction: 300 nsec, laser: 2000 etc.) In order to characterize MalQ overexpressed in E coli, the action pattern of the puri ed enzyme was determined by incubating it with glucose, maltose, maltotriose, maltoheptaose and amylose As shown in Fig 1, there was no any product in the reaction mixture of the enzyme with glucose (Fig 1, lane G1) However, TLC result showed that many longer chain maltodextrins were formed in the enzyme reactions with the other substrates (Fig 1, lane G2, G3 and G7) To understand the e ect of substrate size on the formation of maltodextrin, the reaction mixtures of MalQ with maltose, maltotriose, and maltoheptaose were further analyzed by HPAEC e transfer linear products of G2, G3, and G7 substrates with MalQ ranged DP up to approximately DP12, DP20, and DP35, respectively (Fig 2) It indicated that the larger maltooligosaccharides were treated with MalQ, the longer maltodextrins were brought about Consequently, maltose was the smallest substrate for MalQ reaction MalQ was able to catalyze the reaction of intermolecular transglycosylation in which the enzyme transferred a non-reducing dextrinyl moiety onto another maltodextrin forming their transglycosylation products (Critchley et al., 2001) Figure TLC analysis of reaction pattern of MalQ with glucose (G1), maltose (G2), maltotriose (G3), maltoheptaose (G7), amylose (Aml) Figure HPAEC analysis of side chain distribution of long maltooligosaccharides formed by MalQ from maltose (A), maltotriose (B), and maltoheptaose (C) 84 Journal of Vietnam Agricultural Science and Technology - No.1(3)/2018 TLC plate, the spots of the reaction products did not appear due to the low sensitivity of this method and also indicated amylose was not a preferred substrate for intermolecular transglycosylation of MalQ Intermolecular transglycosylation When amylose was incubated with MalQ, the TLC plate also did not show any product in the reaction mixture (Fig 1, line Aml) However, to rm this result and understand role of glucose, amylose was used as a donor and reacted with 14C-labeled glucose as an acceptor in present of MalQ e chromatogram in Figure was visualized by the naphthol-H2SO4 method and autoradiography (Fig 3, A and B, respectively) e autoradiogram showed a series of transfer short maltooligosaccharides was produced is result indicated that glucose was not the MalQ substrate but able to serves as a high-quality acceptor in the MalQ reaction On the naphtol-H2SO4 treated Tarada et al (1999) improved that the reaction between amylose and amylomaltase from ermus aquaticus produced the cyclic form products To see if MalQ catalyzes the cyclization of amylose, 0.5% (w/v) type III amylose was incubated with the enzyme in the proper bu er, pH and temperature conditions (Tran et al., 2011) e result of HPAEC analysis showed chromatogram pattern at retention time 22 to 38 might be cyclic products (Fig 4A) e aliquot was incubated with β-amylase from barley (10 U/gram of substrate) for hrs at 30°C in TrisHCl pH 7.5 to rm that cycloamylose was present in the reaction mixture A er enzyme treatment, it demonstrated almost the linear glucans were removed and a high maltose contain was produced simultaneously (Fig 4B) e β-amylase-resistant glucans were then separated by double volumes of 99,5% ethanol precipitation and analyzed again by HPAEC e products were eluted at retention time for approximately 22 to 38 min, which was identical to the elution time of cycloamylose (Fig 4C) e molecular weights of the β-amylase-resistant glucans were measured using MALDI-TOF MS (Fig 5) e mixture of the β-amylase-resistant glucans ranged from DP20 to 33 with DP24 as the main product of intermolecular transglycosylation that o en referred to as a coupling reaction of MalQ Figure HPAEC analysis of cyclo-amylose product (A) Reaction mixture of amylose was treated with MalQ; (B) e reaction mixture a er β-amylase treatment; (C) Cycloamylose products Figure MALDI-TOF MS analysis for cyclo-amylose produced by MalQ Figure TLC analysis of reaction pattern generated by MalQ from amylose containing a 14C-labeled glucose e chromatogram was the visualized using naphtol-H2SO4 method (A) and autoradiography (B) Lane 1: the compound of substrate before reaction; Lane 2: the transfer products a er reaction 85 Vietnam Academy of Agricultural Sciences (VAAS) CONCLUSIONS e MalQ, 4-α-glucanotransferase protein of E coli K12, carried out the reaction of disproportionation by intermolecular transglycosylation with maltose as smallest substrate to produce longer maltodextrins However, the molecular size of maltodextrins likely depends on the presence of appropriate acceptor molecules When glucose worked as an acceptor, it led to the production of short matodextrins e enzyme also catalyzed the intermolecular transglycosylation to form cycloamyloses Both of MalQ reaction mechanisms were displayed in Figure ese results are basic data for further study and understanding of E coli metabolism in vivo - Intermolecular transglycosylation - Intramolecular translycosylation Figure Reaction mechanisms of MalQ (4-α-glucanotransferase) ACKNOWLEDGMENT The authors thank Dr Park Kwan Hwa - Seoul National University for supporting fund and facilities and Dr Park Jong Tae for supporting technique skills to implement this study REFERENCES Bhuiyan, S H., Kitaoka M and Hayashi K., 2003 A cycloamylose-forming hyperthermostable 4-α-glucanotransferase of Aquifex aeolicus expressed in Escherichia coli J Mol Catal B Enz., 22: 45-53 Binnema, D J and Euverink G J W., 1998 Use of modi ed starch as an agent for forming a thermoreversible gel Patent application WO9815347 Boos, W and Shuman H., 1998 Maltose/maltodextrin system of Escherichia coli: transport, metabolism, and regulation Microbiol Mol Biol Rev., 62 (1): 204-229 Bradford, M., 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem, 72 (1-2): 248-254 86 Buschiazzo, A., Ugalde J E., Guerin M E, Shepard W., Ugalde R A and Alzari P M., 2004 Crystal structure of glycogen synthase: homologous enzymes catalyze glycogen synthesis and degradation e EMBO J., 23: 3196-3205 Critchley, J H., Zeeman S C., Takaha T., Smith A M and Smith S M., 2001 A critical role for disproportionating enzyme in starch breakdown is revealed by a knock-out mutation in Arabidopsis Plant J., 26: 89-100 Jeon, B S., Taguchi H., Sakai H., Ohshima T., Wakagi T and Matsuzawa H., 1997 4-Alphaglucanotransferase from the hyperthermophilic archaeon ermococcus litoralis-enzyme puri cation and characterization, and gene cloning, sequencing and expression in Escherichia coli Eur J Biochem, 248 (1): 171-178 Kaper, T., Talik B., Ettema T J., Bos H., van der Maarel M J E C and Dijkhuizen L., 2005 Amylomaltase of Pyrobaculum aerophilum IM2 produces thermoreversible starch gels Appl Environ Microbiol, 71: 5098-5106 Lee, H S, Auh J H., Yoon H G., Kim M J., Park J H., Hong S S., Kang M H., Kim T J., Moon T W., Kim J.W and Park K H., 2002 Cooperative action of alpha-glucanotransferase and maltogenic amylase for an improved process of isomaltooligosaccharide (IMO) production J Agric Food Chem., 50(10): 2812-2817 Lin, T P and Preiss J., 1988 Characterization of D-enzyme (4-α-glucanotransferase) in Arabidopsis leaf Plant Physiol (Bethesda), 86 (1): 260-265 O’Neill E.C., Stevenson C.E., Tantanarat K., L atousakis D., Donaldson M.I., Rejzek M., Nepogodiev S.A., Limpaseni T., Field R.A., Lawson D.M., Peat S., Whelan W J and Rees W R., 1915 Structural dissection of maltodextrin disproportionation cycle of the arabidopsis plastidial disproportionation enzyme (DPE1) J Biol Chem., 290 (50): 29834-29853 Palmer, T N., Ryman B E and Whelan W J., 1976 e action pattern of amylomaltase from Escherichia coli Eur J Biochem, 69 (1): 105-115 Peat, S., Whelan W J and Rees W R., 1956 e enzymic synthesis and degradation of starch: the disproportionating enzyme of potato J Chem Soc 44-53 Przylas I., Tomoo K., Terada Y., Takaha T., Fujii K., Saenger W and Sträter N., 2000 Crystal structure amylomatase from ermus aquaticus, a glycosyltransferase catalysing the production of large cyclic glucans J Mol Biol., 296 (3): 873-886 Sato Y., Okamoto-Shibayama K and Azuma T., 2013 e malQ gene is essential for starch matebolism Journal of Vietnam Agricultural Science and Technology - No.1(3)/2018 in Streptococcus mutans J Oral Microbiol, 5: 21285-21293 Tachibana, Y., Takeshi T., Shinsuke F., Masahiro T and Tadayuki I., 2000 Acceptor specificity of 4-α-glucanotransferase from Pyrococcus kodakaraensis KOD1, and synthesis of cycloamylose J Biosci Bioeng, 90 (4): 406-409 Takaha, T., Yanase M., Okada S and Smith S.M., 1996 Potato D-enzyme catalyzes the cyclization of amylose to produce cycloamylose, a novel cyclic glucan J Biol Chem., 271 (6): 2902-2908 Tarada, Y., Fujii K, Takaha T and Okada S., 1999 ermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization: production of cycloamylose Appl Environ Microbiol, 65 (3): 910-915 Tran, P.L., Park, K.H., Park J.T., 2011 Cloning malQ from Escherichia coli K12 and determination of optimal reaction condition of MalQ J Sci - Can o University, 17a: 173-180 (in Vietnamese) Tran, P.L., Lee, J.S., Park, K.H., 2014 Experimental evidence for a 9-binding substrate of Bacillus licheniformis thermostable α-amylase FEBS Lett., 588: 620-624 Tran, P.L., Nguyen, D.H.D., Do, V.H., Kim, Y.L., Park, S.H., Yoo, S.H., Lee, S and Kim, Y.R., 2015 Physicochemical properties of native and partially gelatinized high-amylose jacfruit (Artocarpus heterophyllus Lam.) seed starch LWT Food Sci Technol., 62: 1091-1098 Monod and Torriani A.M., 1948 Synthèse d’un polysaccharide de type amidon aux dèpens du maltose, en prèsence d’un extrait enzymatique d’origine bacterienne Comptes Rendus de l’ Acadèmie des Sciences, 277: 240-242 Date received: 1/10/2018 Date reviewed: 15/10/2018 Reviewer: Assoc Prof Dr Nguyen Hoang Anh Date approved for publication: 25/10/2018 EFFICIENT USE OF CROP RESIDUES FOR PRODUCING ENERGY AND ENHANCING SOIL CARBON SEQUESTRATION AS CLIMATE SMART PRACTICES IN RURAL AREAS OF VIETNAM Mai Van Trinh1, Bui i Phuong Loan1, Nguyen Van iet1, Cao Huong Giang*1 Abstract is article presents results of research on the e cient use of crop residues in energy production and land carbon xation in Ha Tinh, Yen Bai and Bac Lieu MHH-IAE 003 was a product designed and tested by IAE, suitable for many kinds of materials such as: rice husk, sawdust, peanut husk, maize corn, wood chips Compared to similar stoves, MHH-IAE 003 stove was more e ective on heat management and air pollution reduction Biochar get high organic carbon and CEC e biochar was applied to soil, combined with changes in mineral fertilizers in experiments in Ha Tinh, Yen Bai, Bac Lieu e experiment in Yen Bai showed that use of 1.5 tons of biochar per hectare increased corn yield and decreased 20% of chemical fertilizer Rice in Bac Lieu also reached the similar results e peanut experiment sites in Ha Tinh, with the same amount of NPK applied but the greater amount of biochar also got more productive than the other formulas In experiments, biochar method led to increase the amount of organic matter Cation exchange capacity (CEC) was proportional to the amount of organic matter Apply biochar increased soil carbon higher than convention is research has initially led to a successful approach to changing farmer’s crop residue treatment methods, using with a new, more e cient and sustainable way of reducing greenhouse gas emissions Keywords: Gasi er, biochar, crop residues, Yen Bai, Ha Tinh, Bac Lieu INTRODUCTION Agricultural activity is known to be the sector most a ected from climate change, is also a large contributor of greenhouse gas emissions (14%) Factors regulating the GHGs emissions from * agricultural activities are many, among others, over use of chemical fertilizer, over irrigation, etc Burning crop residues in the elds is part of the problem, will be increased in the future as modern cooking facilities replace crop residues cooking devices While Institute for Agriculture Environment (IAE), VAAS Corresponding author: Cao Huong Giang Email: caohuonggiang84vn@gmail.com 87 ... cation of action modes of MalQ encoded by the malQ gene from E coli K12 as a test of E coli matodextrin metabolism in vitro MATERIALS AND METHODS Materials Recombinant E coli carrying malQ gene of. .. production of cycloamylose Appl Environ Microbiol, 65 (3): 910-915 Tran, P.L., Park, K.H., Park J.T., 2011 Cloning malQ from Escherichia coli K12 and determination of optimal reaction condition of MalQ... compound of substrate before reaction; Lane 2: the transfer products a er reaction 85 Vietnam Academy of Agricultural Sciences (VAAS) CONCLUSIONS e MalQ, 4-α-glucanotransferase protein of E coli K12,