An In Vitro Enzyme System for the Production of myo-Inositol from Starch

ABSTRACT We developed an in vitro enzyme system to produce myo-inositol from starch. Four enzymes were used, maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), myo-inositol-3-phosphate synthase (MIPS), and inositol monophosphatase (IMPase). The enzymes were thermostable: MalP and PGM from the hyperthermophilic archaeon Thermococcus kodakarensis, MIPS from the hyperthermophilic archaeon Archaeoglobus fulgidus, and IMPase from the hyperthermophilic bacterium Thermotoga maritima. The enzymes were individually produced in Escherichia coli and partially purified by subjecting cell extracts to heat treatment and removing denatured proteins. The four enzyme samples were incubated at 90°C with amylose, phosphate, and NAD+, resulting in the production of myo-inositol with a yield of over 90% at 2 h. The effects of varying the concentrations of reaction components were examined. When the system volume was increased and NAD+ was added every 2 h, we observed the production of 2.9 g myo-inositol from 2.9 g amylose after 7 h, achieving gram-scale production with a molar conversion of approximately 96%. We further integrated the pullulanase from T. maritima into the system and observed myo-inositol production from soluble starch and raw potato with yields of 73% and 57 to 61%, respectively. IMPORTANCE myo-Inositol is an important nutrient for human health and provides a wide variety of benefits as a dietary supplement. This study demonstrates an alternative method to produce myo-inositol from starch with an in vitro enzyme system using thermostable maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), myo-inositol-3-phosphate synthase, and myo-inositol monophosphatase. By utilizing MalP and PGM to generate glucose 6-phosphate, we can avoid the addition of phosphate donors such as ATP, the use of which would not be practical for scaled-up production of myo-inositol. myo-Inositol was produced from amylose on the gram scale with yields exceeding 90%. Conversion rates were also high, producing over 2 g of myo-inositol within 4 h in a 200-ml reaction mixture. By adding a thermostable pullulanase, we produced myo-inositol from raw potato with yields of 57 to 61% (wt/wt). The system developed here should provide an attractive alternative to conventional methods that rely on extraction or microbial production of myo-inositol.

Roles of maltodextrin and glycogen phosphorylases in maltose utilization and glycogen metabolism in Corynebacterium glutamicum

Corynebacterium glutamicum transiently accumulates large amounts of glycogen, when cultivated on glucose and other sugars as a source of carbon and energy. Apart from the debranching enzyme GlgX, which is required for the formation of maltodextrins from glycogen, α-glucan phosphorylases were assumed to be involved in glycogen degradation, forming α-glucose 1-phosphate from glycogen and from maltodextrins. We show here that C. glutamicum in fact possesses two α-glucan phosphorylases, which act as a glycogen phosphorylase (GlgP) and as a maltodextrin phosphorylase (MalP). By chromosomal inactivation and subsequent analysis of the mutant, cg1479 was identified as the malP gene. The deletion mutant C. glutamicum ΔmalP completely lacked MalP activity and showed reduced intracellular glycogen degradation, confirming the proposed pathway for glycogen degradation in C. glutamicum via GlgP, GlgX and MalP. Surprisingly, the ΔmalP mutant showed impaired growth, reduced viability and altered cell morphology on maltose and accumulated much higher concentrations of glycogen and maltodextrins than the wild-type during growth on this substrate, suggesting an additional role of MalP in maltose metabolism of C. glutamicum. Further assessment of enzyme activities revealed the presence of 4-α-glucanotransferase (MalQ), glucokinase (Glk) and α-phosphoglucomutase (α-Pgm), and the absence of maltose hydrolase, maltose phosphorylase and β-Pgm, all three known to be involved in maltose utilization by Gram-positive bacteria. Based on these findings, we conclude that C. glutamicum metabolizes maltose via a pathway involving maltodextrin and glucose formation by MalQ, glucose phosphorylation by Glk and maltodextrin degradation via the reactions of MalP and α-Pgm, a pathway hitherto known to be present in Gram-negative rather than in Gram-positive bacteria.

Glucose- and Glucokinase-Controlled mal Gene Expression in Escherichia coli

ABSTRACTMalT is the central transcriptional activator of allmalgenes inEscherichia coli. Its activity is controlled by the inducer maltotriose. It can be inhibited by the interaction with certain proteins, and its expression can be controlled. We report here a novel aspect ofmalgene regulation: the effect of cytoplasmic glucose and glucokinase (Glk) on the activity and the expression of MalT. Amylomaltase (MalQ) is essential for the metabolism of maltose. It forms maltodextrins and glucose from maltose or maltodextrins. We found that glucose above a concentration of 0.1 mM blocked the activity of the enzyme.malQmutants when grown in the absence of maltodextrins are endogenously induced by maltotriose that is derived from the degradation of glycogen. Therefore, the fact thatglk malQ+mutants showed elevatedmalgene expression finds its explanation in the reduced ability to remove glucose from MalQ-catalyzed maltodextrin formation and is caused by a metabolically induced MalQ−phenotype. However, even in mutants lacking glycogen, Glk controls endogenous induction. We found that overexpressed Glk due to its structural similarity with Mlc, the repressor ofmalT, binds to the glucose transporter (PtsG), releasing Mlc and thus increasingmalTrepression. In addition, even in mutants lacking Mlc (and glycogen), the overexpression ofglkleads to a reduction inmalgene expression. We interpret this repression by a direct interaction of Glk with MalT concomitant with MalT inhibition. This repression was dependent on the presence of either maltodextrin phosphorylase or amylomaltase and led to the inactivation of MalT.

Recombinant production and biochemical characterization of a hyperthermostable α-glucan/maltodextrin phosphorylase fromPyrococcus furiosus

Alpha-glucan phosphorylase catalyzes the reversible cleavage of α-1-4-linked glucose polymers into α-D-glucose-1-phosphate. We report the recombinant production of an α-glucan/maltodextrin phosphorylase (PF1535) from a hyperthermophilic archaeon,Pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. The apparent 98 kDa recombinant enzyme was active over a broad range of temperatures and pH, with optimal activity at 80 °C and pH 6.5–7. This archaeal protein retained its complete activity after 24 h at 80 °C in Tris-HCl buffer. Unlike other previously reported phosphorylases, the Ni-affinity column purified enzyme showed broad substrate specificity in both the synthesis and degradation of maltooligosaccharides. In the synthetic direction of the enzymatic reaction, the lowest oligosaccharide required for the chain elongation was maltose. In the degradative direction, the archaeal enzyme can produce glucose-1-phosphate from maltotriose or longer maltooligosaccharides including both glycogen and starch. The specific activity of the enzyme at 80 °C in the presence of 10 mM maltoheptaose and at 10 mg ml–1glycogen concentration was 52 U mg–1and 31 U mg–1, respectively. The apparent Michaelis constant and maximum velocity for inorganic phosphate were 31 ± 2 mM and 0.60 ± 0.02 mM min–1µg–1, respectively. An initial velocity study of the enzymatic reaction indicated a sequential bi-bi catalytic mechanism. Unlike the more widely studied mammalian glycogen phosphorylase, thePyrococcusenzyme is active in the absence of added AMP.

The Maltodextrin System of Escherichia coli: Metabolism and Transport

ABSTRACT The maltose/maltodextrin regulon of Escherichia coli consists of 10 genes which encode a binding protein-dependent ABC transporter and four enzymes acting on maltodextrins. All mal genes are controlled by MalT, a transcriptional activator that is exclusively activated by maltotriose. By the action of amylomaltase, we prepared uniformly labeled [14C]maltodextrins from maltose up to maltoheptaose with identical specific radioactivities with respect to their glucosyl residues, which made it possible to quantitatively follow the rate of transport for each maltodextrin. Isogenic malQ mutants lacking maltodextrin phosphorylase (MalP) or maltodextrin glucosidase (MalZ) or both were constructed. The resulting in vivo pattern of maltodextrin metabolism was determined by analyzing accumulated [14C]maltodextrins. MalP− MalZ+ strains degraded all dextrins to maltose, whereas MalP+ MalZ− strains degraded them to maltotriose. The labeled dextrins were used to measure the rate of transport in the absence of cytoplasmic metabolism. Irrespective of the length of the dextrin, the rates of transport at a submicromolar concentration were similar for the maltodextrins when the rate was calculated per glucosyl residue, suggesting a novel mode for substrate translocation. Strains lacking MalQ and maltose transacetylase were tested for their ability to accumulate maltose. At 1.8 nM external maltose, the ratio of internal to external maltose concentration under equilibrium conditions reached 106 to 1 but declined at higher external maltose concentrations. The maximal internal level of maltose at increasing external maltose concentrations was around 100 mM. A strain lacking malQ, malP, and malZ as well as glycogen synthesis and in which maltodextrins are not chemically altered could be induced by external maltose as well as by all other maltodextrins, demonstrating the role of transport per se for induction.

The Maltodextrin System of Escherichia coli: Glycogen-Derived Endogenous Induction and Osmoregulation

ABSTRACT Strains of Escherichia coli lacking MalQ (maltodextrin glucanotransferase or amylomaltase) are endogenously induced for the maltose regulon by maltotriose that is derived from the degradation of glycogen (glycogen-dependent endogenous induction). A high level of induction was dependent on the presence of MalP, maltodextrin phosphorylase, while expression was counteracted by MalZ, maltodextrin glucosidase. Glycogen-derived endogenous induction was sensitive to high osmolarity. This osmodependence was caused by MalZ. malZ, the gene encoding this enzyme, was found to be induced by high osmolarity even in the absence of MalT, the central regulator of all mal genes. The osmodependent expression of malZ was neither RpoS nor OmpR dependent. In contrast, the malPQ operon, whose expression was also increased at a high osmolarity, was partially dependent on RpoS. In the absence of glycogen, residual endogenous induction of the mal genes that is sensitive to increasing osmolarity can still be observed. This glycogen-independent endogenous induction is not understood, and it is not affected by altering the expression of MalP, MalQ, and MalZ. In particular, its independence from MalZ suggests that the responsible inducer is not maltotriose.