JP6033621B2 - Oligosaccharide synthase and method for producing β-1,2-mannobiose and its derivatives - Google Patents

Description

  The present invention relates to β-1,2, which is a core skeleton of O antigen of pathogenic bacteria, using a newly discovered oligosaccharide synthase β-1,2-mannobiose phosphorylase and an oligosaccharide synthesis reaction catalyzed by the enzyme. -It relates to a method for producing mannobiose and its derivatives.

  In recent years, attention has been focused on the importance of sugar chains of glycoproteins for biological recognition (cell adhesion, antigen-antibody reaction, information transmission, virus infection, etc.). Sugar chains are said to be the third chain after nucleic acids and proteins, and their functions have been elucidated rapidly in recent years. Furthermore, there is an urgent need to elucidate in vivo sugar chain recognition in the sugar chain engineering and sugar chain regenerative medicine research fields.

  However, preparation of a sugar chain sample with a very small amount of expression in a living body has to rely on an organic synthesis method at present, and this difficulty has hindered progress in the field of sugar chain engineering research and sugar chain regenerative medicine. For example, β-1,2-mannooligosaccharide, which is the core skeleton of the O antigen of pathogenic bacteria that cause opportunistic infections such as pneumonia and candidiasis, has been conventionally produced by a complicated multi-step reaction by an organic synthesis method ( Non-Patent Document 1), there is a problem that it is very expensive because efficient large-scale preparation is difficult. Therefore, establishment of a simple method for producing a sugar chain that is a biorecognizable molecular element is urgently needed.

Dromer, F.M. Et al, Antimicrobial Agents and Chemotherapy 46 (12): 3869-3876 (2002).

  Therefore, in view of the above problems, the present invention provides β-1,2-mannobiose, which is a core skeleton of O side chain polysaccharide (O antigen) of pathogenic bacteria that efficiently cause opportunistic infections such as pneumonia and candidiasis, and derivatives thereof. It aims to manufacture.

  As a result of earnest studies to achieve the above-mentioned problems, β-1,2, which is the core skeleton of the O antigen of pathogenic bacteria, using an oligosaccharide synthesis reaction catalyzed by newly discovered β-1,2-mannobiose phosphorylase. -Mannobiose and its derivatives can be produced in one step, and it has been found that mass production by scaling up the reaction system is possible, and the present invention has been completed.

That is, the present invention uses oligosaccharide synthase β-1,2-mannobiose phosphorylase having the following enzymatic properties .
a) Action It acts on α-D-mannose 1-phosphate and D-mannose, D-arabinose, D-lyxose, D-allose, D-ribose, L-rhamnose, D-fructose or D-altrose. Β-1,2-mannobiose, D-mannosyl-β-1,4-D-arabinose, D-mannosyl-β-1,2-D-lyxose, D-mannosyl-β-1,3-D- Allose, D-mannosyl-β-1,3-D-ribose, D-mannosyl-β-1,3-L-rhamnose, D-mannosyl-β-1,5-D-fructose, D-mannosyl-β- Producing 1,1-D-fructose, D-mannosyl-β-1,2-D-altrose or D-mannosyl-β-1,4-D-altrose;
b) Substrate specificity To α-D-mannose 1-phosphate and D-mannose, D-arabinose, D-lyxose, D-allose, D-ribose, L-rhamnose, D-fructose or D-altrose Act;
c) Optimum pH
PH 30 at 30 ° C .;
d) Temperature stability Stable up to 50 ° C. under the condition of pH 6.0;
e) pH stability Stable at pH 5.5-9.0 under conditions of 4 ° C. and 24 hours.

And the manufacturing method of (beta) -1,2-mannobiose of this invention and its derivative is (alpha) -D-mannose 1-phosphate, D-mannose, D-arabinose, D-lyxose, D-allose, D-ribose. Performing an oligosaccharide synthesis reaction in a solution containing L-rhamnose, D-fructose or D-altrose and the oligosaccharide synthase β-1,2-mannobiose phosphorylase; -Mannobiose, D-mannosyl-β-1,4-D-arabinose, D-mannosyl-β-1,2-D-lyxose, D-mannosyl-β-1,3-D-allose, D-mannosyl-β -1,3-D-ribose, D-mannosyl-β-1,3-L-rhamnose, D-mannosyl-β-1,5-D-fructose, D-mannosyl-β-1,1-D-fur It comprises the step of recovering fructose, D-mannosyl-β-1,2-D-altrose or D-mannosyl-β-1,4-D-altrose .

  In addition, the method for producing the β-1,2-mannobiose and derivatives thereof is characterized in that the solution has a pH of 5.0 to 7.0.

  According to the present invention, α-D-mannose 1-phosphate and eight types of monosaccharides (D-mannose, D-arabinose, etc.) are synthesized by an oligosaccharide synthesis reaction catalyzed by β-1,2-mannobiose phosphorylase. D-lyxose, D-allose, D-ribose, L-rhamnose, D-fructose or D-altrose) can be used as a starting material to easily produce β-1,2-mannobiose and its derivatives in one step. it can.

  According to the method of the present invention, β-1,2-mannobiose and its derivatives can be easily produced by an oligosaccharide synthesis reaction which is the reverse reaction of the phosphorolysis reaction.

  Specifically, by performing an oligosaccharide synthesis reaction in a solution containing α-D-mannose 1-phosphate, D-mannose, and β-1,2-mannobiose phosphorylase, β-1,2 -Mannobiose can be produced.

  Moreover, by performing an oligosaccharide synthesis reaction in a solution containing α-D-mannose 1-phosphate, D-arabinose, and β-1,2-mannobiose phosphorylase, D-mannosyl-β-1, 4-D-arabinose can be produced.

  In addition, by performing an oligosaccharide synthesis reaction in a solution containing α-D-mannose 1-phosphate, D-lyxose, and β-1,2-mannobiose phosphorylase, D-mannosyl-β-1, 2-D-lyxose can be produced.

  In addition, by performing an oligosaccharide synthesis reaction in a solution containing α-D-mannose 1-phosphate, D-allose, and β-1,2-mannobiose phosphorylase, D-mannosyl-β-1, 3-D-allose can be produced.

  In addition, by performing an oligosaccharide synthesis reaction in a solution containing α-D-mannose 1-phosphate, D-ribose, and β-1,2-mannobiose phosphorylase, D-mannosyl-β-1, 3-D-ribose can be produced.

  In addition, by performing an oligosaccharide synthesis reaction in a solution containing α-D-mannose 1-phosphate, L-rhamnose, and β-1,2-mannobiose phosphorylase, D-mannosyl-β-1, 3-L-rhamnose can be produced.

  Further, by performing an oligosaccharide synthesis reaction in a solution containing α-D-mannose 1-phosphate, D-fructose, and β-1,2-mannobiose phosphorylase, D-mannosyl-β-1, 5-D-fructose and D-mannosyl-β-1,1-D-fructose can be produced.

  In addition, by performing an oligosaccharide synthesis reaction in a solution containing α-D-mannose 1-phosphate, D-altrose, and β-1,2-mannobiose phosphorylase, D-mannosyl-β-1 , 2-D-altrose and D-mannosyl-β-1,4-D-altrose can be produced.

  The concentration of β-1,2-mannobiose phosphorylase in the reaction solution is not particularly limited, but can be 0.086 to 172 μM, preferably 0.43 to 86 μM.

  Since the optimum pH of β-1,2-mannobiose phosphorylase at 30 ° C. is around 6.0, the solution preferably has a pH of 5.0 to 7.0, particularly preferably 6.0. . The solution is not particularly limited, but a morpholinoethanesulfonic acid-sodium hydroxide (MES-NaOH) buffer is suitable.

  Moreover, since the temperature stability of β-1,2-mannobiose phosphorylase at pH 6.0 is up to 50 ° C., the oligosaccharide synthesis reaction is preferably carried out at 25 to 50 ° C., particularly preferably 30 ° C. .

  The reaction time is not particularly limited, but is preferably 1 to 100 hours, and particularly preferably 24 hours.

  Β-1,2-mannobiose and its derivatives produced by the above oligosaccharide synthesis reaction can be isolated by known methods such as column chromatography and crystallization, but high-performance liquid chromatography is preferred. is there.

  The present invention is not limited to the above embodiment, and various modifications can be made without departing from the spirit of the present invention.

  EXAMPLES Next, although an Example demonstrates this invention in detail, this invention is not limited by these.

  Based on the genomic information of Listeria innocure, a forward primer (SEQ ID NO: 3) and a reverse primer (SEQ ID NO: 4) for the Lin0857 gene were designed and synthesized. The base sequence of the Lin0857 gene is shown in SEQ ID NO: 2, and the amino acid sequence encoded by this base sequence is shown in SEQ ID NO: 1.

  Using Listeria inocure genomic DNA as a template, using the above primer and KOD plus polymerase (manufactured by TOYOBO), holding at 95 ° C. for 2 minutes, followed by 95 ° C. for 30 seconds, 58 ° C. for 30 seconds, 68 ° C. for 1 minute A PCR reaction was performed by repeating a cycle of 30 minutes for 45 seconds 45 times, and finally held at 68 ° C. for 5 minutes. As a result, an amplified fragment of 1084 bp was obtained. The DNA fragment amplified by this PCR is DNA encoding Lin0857 having an NdeI site at the 5 'end and an XhoI site at the 3' end.

  The obtained amplified fragment was digested with restriction enzymes NdeI and XhoI, and then ligated to a commercially treated plasmid for gene expression pET-24a (manufactured by Novagen) using a highly efficient ligation reagent Ligation high (manufactured by TOYOBO). . Furthermore, an Escherichia coli competent cell DH5α (manufactured by TOYOBO) was transformed with the ligation reaction solution, and an expression vector pET-24a containing DNA encoding Lin0857 to which a His tag consisting of 6-residue histidine was added at the C-terminus. Was recovered.

  Using this expression vector pET-24a, E. coli BL21 (DE3) was transformed according to the method of Hanahan et al. (J. Mol. Biol., 1983, 166, 557-580). The transformant was inoculated into 200 mL of LB medium containing 50 μg / mL kanamycin, and induced culture was performed at 18 ° C. for 24 hours with an IPTG concentration of 0.1 mM. The cells recovered from the culture solution by centrifugation are suspended in 10 mL of 20 mM HEPES-NaOH buffer pH 7.5 containing 500 mM sodium chloride and 10% glycerol, disrupted by sonication, and then centrifuged to obtain a crude enzyme solution. Got. The recombinant protein was purified by column chromatography using a His tag protein purification column HisTrapFF (manufactured by GE Healthcare). The obtained purified enzyme solution was dialyzed against 10 mM HEPES-NaOH buffer pH 7.0, and concentrated to 4 mL by ultrafiltration using a centrifugal filter unit Amicon Ultra-15 (Millipore). A purified enzyme preparation was prepared.

  Using the obtained purified enzyme preparation, this protein was identified as a novel enzyme β-1,2-mannobiose phosphorylase by the following method, and β-1,2-mannobiose and nine types of β-1,2 were identified. -Mannobiose derivatives were produced.

  In 1 ml of 40 mM MES-NaOH buffer (pH 6.0) containing 50 mM α-D-mannose 1-phosphate (sugar donor), 50 mM D-mannose (sugar acceptor), 0.43 μM purified enzyme The enzyme reaction was performed at 30 ° C. for 24 hours. The reaction solution was desalted with Amberlite MB3 (manufactured by Organo), and the disaccharide fraction was isolated by high performance liquid chromatography using 70% acetonitrile as a solvent on a TSKgel Amide-80 column (manufactured by TOSOH). The yield after purification was 2 mg. When the product was analyzed by NMR, it was confirmed that it was β-1,2-mannobiose (the following formula (1)).

  50 mM α-D-mannose 1-phosphate (sugar donor), 50 mM sugar acceptors (5 types: D-arabinose, D-lyxose, D-allose, D-ribose, L-rhamnose), 86 μM The enzyme reaction was performed at 30 ° C. for 24 hours in 1 ml of 40 mM MES-NaOH buffer (pH 6.0) containing the purified enzyme. Each reaction solution was desalted with Amberlite MB3, and then a disaccharide fraction was isolated by high performance liquid chromatography using 75% acetonitrile as a solvent on a TSKgel Amide-80 column, and each product was analyzed by NMR. -Mannosyl-β-1,4-D-arabinose (the following formula (2)), D-mannosyl-β-1,2-D-lyxose (the following formula (3)), D-mannosyl-β-1,3 -D-allose (the following formula (4)), D-mannosyl-β-1,3-D-ribose (the following formula (5)), D-mannosyl-β-1,3-L-rhamnose (the following formula ( 6)). Yields after purification were 1 mg, 1 mg, 1 mg, 3 mg and 1 mg, respectively.

  Enzymatic reaction in 1 ml of 40 mM MES-NaOH buffer (pH 6.0) containing 50 mM α-D-mannose 1-phosphate (sugar donor), 50 mM D-fructose (sugar acceptor), 86 μM of purified enzyme Was performed at 30 ° C. for 24 hours. In the same manner as in Example 2, a disaccharide fraction (2 mg) was isolated from each reaction solution, and the product was analyzed by NMR. As a result, when fructose was used as a sugar acceptor and the reaction was performed under the above conditions, the products were D-mannosyl-β-1,5-D-fructose (the following formula (7)) and D-mannosyl-β-1, It confirmed that it was a mixture of 1-D-fructose (following formula (8)). The mixture was separable by high performance liquid chromatography using 80% acetonitrile as a solvent on a Shodex Asahi Pack NH2P-50 4E column.

  In 1 ml of 40 mM MES-NaOH buffer (pH 6.0) containing 50 mM α-D-mannose 1-phosphate (sugar donor), 50 mM D-fructose (sugar acceptor), and 2.9 μM of purified enzyme The enzyme reaction was performed at 30 ° C. for 24 hours. After desalting the reaction solution with Amberlite MB3, the disaccharide fraction (1 mg) was isolated by high-performance liquid chromatography using 80% acetonitrile as a solvent on a Shodex Asahi Pack NH2P-50 4E column, and the product was analyzed by NMR. As a result of analysis, it was confirmed that the product when reacted under the above conditions was only D-mannosyl-β-1,5-D-fructose (the following formula (7)).

  The enzyme reaction was carried out in 1 ml of 40 mM MES-NaOH buffer (pH 6.0) containing 50 mM α-D-mannose 1-phosphate (sugar donor), 50 mM D-altrose (sugar acceptor) and 86 μM of purified enzyme. This was performed at 30 ° C. for 4 hours. As in Example 2, the disaccharide fraction (3 mg) was isolated from each reaction solution, and the product was analyzed by NMR. As a result, the main component of the disaccharide fraction was D-mannosyl-β-1,2-D-. It was altrose (the following formula (9)), and it was confirmed that at least D-mannosyl-β-1,4-D-altrose (the following formula (10)) was mixed as a minor component.

  Using 2 mM α-D-mannose 1-phosphate and D-mannose in 25 mM MES-NaOH buffer (pH 6.0), the phosphoric acid produced during the synthesis reaction was converted to the molybdenum blue method (J. Biol. Chem. 1946, 162, 421-428). The activity to produce 1 μmol of phosphoric acid per minute under the above conditions was defined as 1 unit. As a result, the activity when D-mannose was used as a sugar receptor was 6.1 units / mg.

  The optimum pH of β-1,2-mannobiose phosphorylase at 30 ° C. is around 6.0 (FIG. 1A), and the stable pH range is 5.5 to 9.0 at 4 ° C. and 24 hours. (FIG. 1B). The stability of the enzyme at pH 6.0 was up to 50 ° C. (FIG. 1C).

  As described above, the present invention can be used in the sugar chain engineering research field and the sugar chain medical industry.

FIG. 2 is a view showing the optimum pH of β-1,2-mannobiose phosphorylase prepared in Example 1. FIG. 2 is a view showing the pH stability of β-1,2-mannobiose phosphorylase prepared in Example 1. FIG. 3 is a graph showing the temperature stability of β-1,2-mannobiose phosphorylase prepared in Example 1.

Claims (2)

α-D-mannose 1-phosphate, D-mannose, D-arabinose, D-lyxose, D-allose, D-ribose, L-rhamnose, D-fructose or D-altrose and the following enzymatic A step of performing an oligosaccharide synthesis reaction in a solution containing an oligosaccharide synthase β-1,2-mannobiose phosphorylase having properties , β-1,2-mannobiose, D-mannosyl-β-1,4- D-arabinose, D-mannosyl-β-1,2-D-lyxose, D-mannosyl-β-1,3-D-allose, D-mannosyl-β-1,3-D-ribose, D-mannosyl- β-1,3-L-rhamnose, D-mannosyl-β-1,5-D-fructose, D-mannosyl-β-1,1-D-fructose, D-mannosyl-β-1,2-D- Alto A method for producing β-1,2-mannobiose and derivatives thereof, comprising a step of recovering sucrose or D-mannosyl-β-1,4-D-altrose :
a) Action
acting on α-D-mannose 1-phosphate and D-mannose, D-arabinose, D-lyxose, D-allose, D-ribose, L-rhamnose, D-fructose or D-altrose; -1,2-mannobiose, D-mannosyl-β-1,4-D-arabinose, D-mannosyl-β-1,2-D-lyxose, D-mannosyl-β-1,3-D-allose, D -Mannosyl-β-1,3-D-ribose, D-mannosyl-β-1,3-L-rhamnose, D-mannosyl-β-1,5-D-fructose, D-mannosyl-β-1,1 Producing D-fructose, D-mannosyl-β-1,2-D-altrose or D-mannosyl-β-1,4-D-altrose;
b) Substrate specificity
acts on α-D-mannose 1-phosphate and D-mannose, D-arabinose, D-lyxose, D-allose, D-ribose, L-rhamnose, D-fructose or D-altrose;
c) Optimum pH
PH 30 at 30 ° C .;
d) Temperature stability
stable to 50 ° C. under pH 6.0 conditions;
e) pH stability
Stable at pH 5.5-9.0 under conditions of 4 ° C. and 24 hours. The solution, beta-1,2-mannobiose and a manufacturing method of a derivative according to claim 1, characterized in that the pH 5.0-7.0.

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