Α-glucan Phosphorylase Research Articles

The plastidial glucan phosphorylase modulates maltodextrin metabolism and affects starch parameters in Arabidopsis thaliana.

The plastidial α-glucan phosphorylase (PHS1) can catalyze the elongation and degradation of glucans, but its exact physiological role in plants is not completely deciphered. A plethora of studies have indicated that PHS1 is involved in transitory starch turnover, both in photosynthetic tissues as well as reserve starch accumulation in sink organs of multiple species, by exerting its effects on the plastidial maltodextrin pools. Recent studies have also established its role in the mobilization of short maltooligosaccharides (MOSs), thereby assisting in starch granule initiation. This paper highlights our current findings from studying four constitutive double-knock out mutants related to the plastidial maltodextrin metabolism namely phs1dpe1, phs1ptst2, phs1pgm1 and phs1isa3 of Arabidopsis thaliana. Our studies have indicated different effects on carbon partitioning in these double mutants. The carbon allocation between starch and sucrose in different double mutants varied with respect to time and light conditions with significant overall changes in phs1dpe1. Furthermore, a potential time specific function of PHS1 in maltodextrin metabolism has come to light. Changes in maltodextrin turnover exerted effects on the starch granule number and size in the double mutants especially phs1dpe1. The characterized double mutants have been further assessed in terms of photosynthetic efficiency, and starch parameters such as internal structure and morphology in detail. Our results have shown that the different photosynthetic parameters in pgm1 and its corresponding double mutant were affected relative to WT and phs1. However, other double mutants were not impaired in terms of photosynthetic efficiency despite alterations in their MOS levels.

Combining an Enhanced Polyphosphate Kinase-Driven UDP-Glucose Regeneration System with the Screening of Key Glycosyltransferases for Efficient In Vitro Synthesis of Nucleoside Disaccharides.

Nucleoside disaccharides are essential glycosides that naturally occur in specific living organisms. This study developed an enhanced UDP-glucose regeneration system to facilitate the in vitro multienzyme synthesis of nucleoside disaccharides by integrating it with nucleoside-specific glycosyltransferases. The system utilizes maltodextrin and polyphosphate as cost-effective substrates for UDP-glucose supply, catalyzed by α-glucan phosphorylase (αGP) and UDP-glucose pyrophosphorylase (UGP). To address the low activity of known polyphosphate kinases (PPKs) in the UDP phosphorylation reaction, a sequence-driven screening identified RhPPK with high activity against UDP (>1000 U/mg). Computational design further led to the creation of a double mutant with a 2566-fold increase in thermostability at 50 °C. The enhanced UDP-glucose regeneration system increased the production rate of nucleoside disaccharide synthesis by 25-fold. In addition, our UDP-glucose regeneration system is expected to be applied to other glycosyl transfer reactions.

Initiation of B-type starch granules in wheat endosperm requires the plastidial α-glucan phosphorylase PHS1.

The plastidial α-glucan phosphorylase (PHS1) can elongate and degrade maltooligosaccharides (MOSs), but its exact physiological role in plants is poorly understood. Here, we discover a specialized role of PHS1 in establishing the unique bimodal characteristic of starch granules in wheat (Triticum spp.) endosperm. Wheat endosperm contains large A-type granules that initiate at early grain development and small B-type granules that initiate in later grain development. We demonstrate that PHS1 interacts with B-GRANULE CONTENT1 (BGC1), a carbohydrate-binding protein essential for normal B-type granule initiation. Mutants of tetraploid durum wheat (Triticum turgidum) deficient in all homoeologs of PHS1 had normal A-type granules but fewer and larger B-type granules. Grain size and starch content were not affected by the mutations. Further, by assessing granule numbers during grain development in the phs1 mutant and using a double mutant defective in both PHS1 and BGC1, we demonstrate that PHS1 is exclusively involved in B-type granule initiation. The total starch content and number of starch granules per chloroplast in leaves were not affected by loss of PHS1, suggesting that its role in granule initiation in wheat is limited to the endosperm. We therefore propose that the initiation of A- and B-type granules occurs via distinct biochemical mechanisms, where PHS1 plays an exclusive role in B-type granule initiation.

Analysis of the Differentially Expressed Proteins and Metabolic Pathways of Honeybush (Cyclopia subternata) in Response to Water Deficit Stress.

Honeybush (Cyclopia spp.) is a rich source of antioxidant properties and phenolic compounds. Water availability plays a crucial role in plant metabolic processes, and it contributes to overall quality. Thus, this study aimed to investigate changes in molecular functions, cellular components, and biological processes of Cyclopia subternata exposed to different water stress conditions, which include well-watered (as Control, T1), semi-water stressed (T2), and water-deprived (T3) potted plants. Samples were also collected from a well-watered commercial farm first cultivated in 2013 (T13) and then cultivated in 2017 (T17) and 2019 (T19). Differentially expressed proteins extracted from C. subternata leaves were identified using LC-MS/MS spectrometry. A total of 11 differentially expressed proteins (DEPs) were identified using Fisher's exact test (p < 0.00100). Only α-glucan phosphorylase was found to be statistically common between T17 and T19 (p < 0.00100). Notably, α-glucan phosphorylase was upregulated in the older vegetation (T17) and downregulated in T19 by 1.41-fold. This result suggests that α-glucan phosphorylase was needed in T17 to support the metabolic pathway. In T19, five DEPs were upregulated, while the other six were downregulated. Based on gene ontology, the DEPs in the stressed plant were associated with cellular and metabolic processes, response to stimulus, binding, catalytic activity, and cellular anatomical entity. Differentially expressed proteins were clustered based on the Kyoto Encyclopedia of Genes and Genomes (KEGG), and sequences were linked to metabolic pathways via enzyme code and KEGG ortholog. Most proteins were involved in photosynthesis, phenylpropanoid biosynthesis, thiamine, and purine metabolism. This study revealed the presence of trans-cinnamate 4-monooxygenase, an intermediate for the biosynthesis of a large number of substances, such as phenylpropanoids and flavonoids.

Pho1 cooperates with DPE1 to control short maltooligosaccharide mobilization during starch synthesis initiation in rice endosperm.

Plastidial α-glucan phosphorylase is a key factor that cooperates with plastidial disproportionating enzyme to control short maltooligosaccharide mobilization during the initiation process of starch molecule synthesis in developing rice endosperm. Storage starch synthesis is essential for grain filling. However, little is known about how cereal endosperm controls starch synthesis initiation. One of core events for starch synthesis initiation is short maltooligosaccharide (MOS) mobilization consisting of long MOS primer production and excess MOS breakdown. By mutant analyses and biochemical investigations, we present here functional identifications of plastidial α-glucan phosphorylase (Pho1) and disproportionating enzyme (DPE1) during starch synthesis initiation in rice (Oryza sativa) endosperm. Pho1 deficiency impaired MOS mobilization, triggering short MOS accumulation and starch synthesis reduction during early seed development. The mutant seeds differed significantly in MOS level and starch content at 15days after flowering and exhibited diverse endosperm phenotypes during mid-late seed development: ranging from pseudonormal to shrunken (Shr), severely or excessively Shr. The level of DPE1 was almost normal in the PN seeds but significantly reduced in the Shr seeds. Overexpression of DPE1 in pho1 resulted in plump seeds only. DPE1 deficiency had no obvious effects on MOS mobilization. Knockout of DPE1 in pho1 completely blocked MOS mobilization, resulting in severely and excessively Shr seeds only. These findings show that Pho1 cooperates with DPE1 to control short MOS mobilization during starch synthesis initiation in rice endosperm.

Biosynthesis of artificial starch and microbial protein from agricultural residue

Growing populations and climate change pose great challenges to food security. Humankind is confronting a serious question: how will we feed the world in the near future? This study presents an out-of-the-box solution involving the highly efficient biosynthesis of artificial starch and microbial proteins from available and abundant agricultural residue as new feed and food sources. A one-pot biotransformation using an in vitro coenzyme-free synthetic enzymatic pathway and baker's yeast can simultaneously convert dilute sulfuric acid-pretreated corn stover to artificial starch and microbial protein under aerobic conditions. The β-glucosidase-free commercial cellulase mixture plus an ex vivo two-enzyme complex containing cellobiose phosphorylase and potato α-glucan phosphorylase displayed on the surface of Saccharomyces cerevisiae, showed better cellulose hydrolysis rates than a commercial β-glucosidase-rich cellulase mixture. This is because the channeling of the hydrolytic product from the solid cellulosic feedstock to the yeast mitigated the inhibition of the cellulase cocktail. Animal tests have shown that the digestion of artificial amylose results in slow and relatively small changes in blood sugar levels, suggesting that it could be a new health food component that prevents obesity and diabetes. A combination of the utilization of available agricultural residue and the biosynthesis of starch and microbial protein from non-food biomass could address the looming food crisis in the food-energy-water nexus.

The Plastidial Glucan Phosphorylase Affects the Maltooligosaccharide Metabolism in Parenchyma Cells of Potato (Solanum tuberosum L.) Tuber Discs.

Maltodextrin metabolism is thought to be involved in both starch initiation and degradation. In this study, potato tuber discs from transgenic lines containing antisense constructs against the plastidial and cytosolic isoforms of α-glucan phosphorylase and phosphoglucomutase were used to evaluate their influences on the conversion of externally supplied glucose-1-phosphate into soluble maltodextrins, as compared to wild-type potato tubers (Solanum tuberosum L. cv. Desiree). Relative maltodextrin amounts analyzed by capillary electrophoresis with laser-induced fluorescence revealed that tuber discs could immediately uptake glucose-1-phosphate and use it to produce maltooligosaccharides with a degree of polymerization of up to 30, as opposed to tubers repressing the plastidial glucan phosphorylase. The results presented here support previous indications that a specific transporter for glucose-1-phosphate may exist in both the plant cells and the plastidial membranes, thereby allowing a glucose-6-phosphate-independent transport. Furthermore, it confirms that the plastidial glucan phosphorylase is responsible for producing longer maltooligosaccharides in the plastids by catalyzing a glucosyl polymerization reaction when glucose-1-phosphate is available. All these findings contribute to a better understanding of the role of the plastidial phosphorylase as a key enzyme directly involved in the synthesis and degradation of glucans and their implication on starch metabolism.

Effect of Starch Primers on the Fine Structure of Enzymatically Synthesized Glycogen-like Glucan.

Glycogen-like glucan (GnG) is a unique hyperbranched polysaccharide nanoparticle which is drawing increasing attention due to its biodegradability and abundant short branches that can be functionalized. Because starch and GnG are both composed of glucose residues and have similar glucosidic bonds, GnG could be fabricated by sucrose phosphorylase, α-glucan phosphorylase, and branching enzymes from starch primers and sucrose. In this study, high-amylose starch, normal starch, and waxy corn starch were used as primers to synthesize GnG, and their impact on the fine structure of GnG was investigated. Structural analysis indicated that with increasing content of amylopectin in the starch primer, the proportion of short chains in GnG decreased, and the degree of β-amylolysis and α-amylolysis was enhanced. Amylose in the primer contributed to a compact and homogeneous structure of GnG, while amylopectin triggered the formation of branch points with a more open distribution. These findings provide a new strategy for regulating the fine structure of GnG.

Comparative analysis of anatomical structure, assimilate accumulation and gene expression in lignin and carbohydrate metabolism pathway during taproot thickening of taicai and pak choi

• Taicai taproots have a more scattered distribution of xylem vessels than pak choi taproots. • Taicai taproots accumulate more sucrose and amylopectin than pak choi taproots. • The genes encoding α-glucan phosphorylase 2 ( PHS2 ) and glucan-6-phosphate/phosphate translocator 2 ( GPT2 ) probably play a major role in starch accumulation in taicai taproots. • Transcription factors from four families probably regulate carbohydrate accumulation and cell proliferation in taicai taproots. Taicai ( Brassica rapa L.ssp. chinensis Makino var. tai-tsai Hort) is a type of brassica vegetable that can form edible fleshy roots, and is a variant of pak choi ( Brassica rapa L. ssp. chinensis (L.) Hanelt). Its expanded taproot is an important nutrient storage organ. To better understand the changes and their regulation during taproot formation in taicai, we compared and analyzed the anatomical structure, lignin synthesis, carbohydrate accumulation and mRNA expression profile in the axial roots of taicai and pak choi, respectively. Compared with pak choi, the taproot of taicai had more developed phloem, a more scattered distribution of xylem vessels, and more sucrose and amylopectin accumulation. Comparison results based on RNA-seq data and qRT-PCR also reflected a greater reduction in lignin synthesis in taicai. Two genes encoding α-glucan phosphorylase 2 ( BrPHS2 ) and glucan-6-phosphate/phosphate translocator 2 ( BrGPT2 ) were suggested to play a noteworthy role in the starch accumulation during the taproot formation in taicai. Weighted gene co-expression network analysis (WGCNA) analysis identified two modules highly associated with the increase of root diameter; these modules contain genes related to sucrose starch metabolism as well as cell proliferation. Several transcription factors from four families in the module may regulate the genes related to carbohydrate metabolism and cell development in taicai taproot. These results contributed to a better understanding of the differences in root development among brassicas, and provide valuable resources for further exploring the regulation of the formation of modified organ in brassicas.

Enhanced biosynthesis of d-tagatose from maltodextrin through modular pathway engineering of recombinant Escherichia coli

In our previous study, a whole-cell biocatalytic system involving α-glucan phosphorylase, phosphoglucomutase, glucose 6-phosphate isomerase, tagatose 1,6-bisphosphate aldolase, and phosphoglycolate phosphatase, was constructed to produce d-tagatose from maltodextrin. However, the biotransformation efficiency of this whole-cell catalyst was low because of the unbalanced ratio of each enzyme and metabolic flux of the intermediates. Therefore, in this study, the biosynthesis of d-tagatose was enhanced by optimizing vectors and improving the expression of rate-limiting enzymes through the construction of multi-copy genes to regulate expression levels. The conversion rate increased from 20.8% after 24 h to 25.2% after 3 h using 10 g/L maltodextrin as the substrate. Furthermore, the genes in the bypass pathways in Escherichia coli, pfka and zwf, were deleted to increase the accumulation of intermediates. The strain ER-2GatZ (ΔpΔz) produced 3.383 g/L d-tagatose using 10 g/L substrate after 3 h, which was 1.34 times that of the wild strain. This work exemplifies the use of modular engineering to enhance d-tagatose production using whole-cell catalysts.

Controlling the Fine Structure of Glycogen-like Glucan by Rational Enzymatic Synthesis.

Glycogen-like glucan (GnG), a hyperbranched glucose polymer, has been receiving increasing attention to generate synthetic polymers and nanoparticles. Importantly, different branching patterns strongly influence the functionality of GnG. To uncover ways of obtaining different GnG branching patterns, a series of GnG with radius from 22.03 to 27.06 nm were synthesized using sucrose phosphorylase, α-glucan phosphorylase (GP), and branching enzyme (BE). Adjusting the relative activity ratio of GP and BE (GP/BE) made the molecular weight (MW) distribution of intermediate GnG products follow two different paths. At a low GP/BE, the GnG developed from "small to large" during the synthetic process, with the MW increasing from 6.15 × 106 to 1.21 × 107 g/mol, and possessed a compact structure. By contrast, a high GP/BE caused the "large to small" model, with the MW reduction of GnG from 1.62 × 107 to 1.21 × 107 g/mol, and created a loose external structure. The higher GP activity promoted the elongation of external chains and restrained chain transfer by the BE to the inner zone of GnG, which would modulate the loose-tight structure of GnG. These findings provide new useful insights into the construction of structurally well-defined nanoparticles.

Dictyoglomus turgidum DSM 6724 α-Glucan Phosphorylase: Characterization and Its Application in Multi-enzyme Cascade Reaction for D-Tagatose Production.

Phosphorylase is a type of enzyme-producing sugar phosphates through the reversible phosphorolysis reactions of glycosides, which makes it an important starting enzyme in multi-enzyme systems for rare sugar biomanufacturing. To investigate its application in D-tagatose biosynthesis from maltodextrin using in vitro multi-enzyme cascade biosystem, the α-glucan phosphorylase (αGP; EC 2.4.1.1) from the thermophile D. turgidum DSM 6724 was prepared and characterized. It exhibited the specific activity of 30.28 U/mg at its optimal temperature of 70°C. Thermostability results revealed that DituαGP could maintain more than 25% of initial activity for 4h, even at 90°C. The highest activity was observed at pH 5.5, and most divalent metal ions deactivated the enzyme. DituαGP exhibited great application potential in the multi-enzyme system that about 3.919g/L of D-tagatose was produced from 150g/L of maltodextrin within 36h. DituαGP has played an important role in this biosystem and will also be applied in the synthesis of other rare sugars from maltodextrin.

Thermostable α-Glucan Phosphorylase-catalyzed Enzymatic Chain-elongation to Produce 6-Deoxygenated α(1γ4)-Oligoglucans

: Herein, the synthesis of 6-deoxygenated α(1γ4)-oligoglucans via thermostable αglucan phosphorylase (from Aquifex aeolicus VF5)-catalyzed enzymatic chain-elongation is reported from a maltotriose primer using a non-native substrate, 6-deoxy-α-D-glucose 1- phosphate. The enzymatic reaction of the two substrates mainly produced a tetrasaccharide with one 6-deoxy-α-D-glucose unit at the non-reducing end, together with a minor pentasaccharide with two 6-deoxy-α-D-glucose units. The enzymatic chain-elongation from the primer in the presence of 6-deoxy-α-D-glucose 1-phosphate and a native substrate, α-D-glucose 1- phosphate, afforded 6-deoxygenated α(1γ4)-oligoglucans with varying degrees of polymerization. This enzymatic chain-elongation catalyzed by thermostable α-glucan phosphorylase is an efficient method to produce non-natural oligosaccharides, that is, 6-deoxygenated α(1γ4)- oligoglucans.

Facile biosynthesis of synthetic crystalline cellulose nanoribbon from maltodextrin through a minimized two-enzyme phosphorylase cascade and its application in emulsion

Nanocellulose has many promising applications such as a green ingredient for Pickering emulsion. Traditional strategies to produce nanocellulose, which are acid or enzymatic hydrolysis and mechanical methods on natural complicated cellulose, are hard to control and can result in significant pollutants during the processes. Herein, we demonstrated a facile and sustainable method for the biocatalytic production of insoluble synthetic crystalline cellulose nanoribbon (CCNR) from cheap maltodextrin by coupling α-glucan phosphorylase (αGP) and cellodextrin phosphorylase (CDP) using cellobiose as a primer. And by optimizing the combination of different αGP and CDP, it turned out that the optimal enzyme combination is αGP from Thermotoga maritime and CDP from Clostridium thermocellum, in which CDP was attached to a family 9 cellulose-binding module. The product yield and degree of polymerization (DP) of insoluble synthetic CCNR was affected by the primer concentration at a fixed concentration of maltodextrin. After optimization of reaction conditions, the highest product yield of insoluble synthetic CCNR was 44.92 % and the highest DP of the insoluble synthetic CCNR was 24 from 50 g 1-1 maltodextrin. This insoluble synthetic CCNR can be used as a Pickering emulsions stabilizer, showing excellent emulsifiability. This study provides a promising alternative for cost-efficient production of insoluble synthetic CCNR which was used as a green emulsion stabilizer.

Phosphorolytic degradation of leaf starch via plastidic α-glucan phosphorylase leads to optimized plant growth and water use efficiency over the diel phases of Crassulacean acid metabolism.

In plants with Crassulacean acid metabolism (CAM), it has been proposed that the requirement for nocturnal provision of phosphoenolpyruvate as a substrate for CO2 uptake has resulted in a re-routing of chloroplastic starch degradation from the amylolytic route to the phosphorolytic route. To test this hypothesis, we generated and characterized four independent RNAi lines of the obligate CAM species Kalanchoë fedtschenkoi with a >10-fold reduction in transcript abundance of plastidic α-glucan phosphorylase (PHS1). The rPHS1 lines showed diminished nocturnal starch degradation, reduced dark CO2 uptake, a reduction in diel water use efficiency (WUE), and an overall reduction in growth. A re-routing of starch degradation via the hydrolytic/amylolytic pathway was indicated by hyperaccumulation of maltose in all rPHS1 lines. Further examination indicated that whilst operation of the core circadian clock was not compromised, plasticity in modulating net dark CO2 uptake in response to changing photoperiods was curtailed. The data show that phosphorolytic starch degradation is critical for efficient operation of the CAM cycle and for optimizing WUE. This finding has clear relevance for ongoing efforts to engineer CAM into non-CAM species as a means of boosting crop WUE for a warmer, drier future.

Identification of proteins and metabolic networks associated with sucrose accumulation in sugarcane (Saccharum spp. interspecific hybrids)

ABSTRACT The rate of improvement of sugar content in sugarcane remains low for decades worldwide. Our previous transcriptome studies provided an atlas of sucrose accumulation-related gene expression, but little is known about the proteins involved. Here, we conducted a proteomic analysis of experimentally altered sucrose accumulation in sugarcane. Analysis of stem proteomes of sugarcane ripener ethephon treated high- and low-sugar genotypes had identified 2983 proteins of which 139 were significantly differentially expressed (DEPs). These DEPs were found to be associated with sugar metabolism-related processes with 25 of them may have a regulatory role in sucrose accumulation. The key proteins identified include UDP-glucose 6-dehydrogenase associated with amino sugar and nucleotide sugar metabolism; those involved in carbon fixation; and fructokinase, β-D-glucosidase, and α-glucan phosphorylase involved in starch and sucrose metabolism. Distinct genotype- and ethephon-dependent DEP expression was evident providing new insights into one of the most intractable sugarcane traits to breeding.

Thermostable α-Glucan Phosphorylase-Catalyzed Enzymatic Copolymerization to Produce Partially 2-Deoxygenated Amyloses

α-Glucan phosphorylase catalyzes the enzymatic polymerization of α-d-glucose 1-phosphate (Glc-1-P) monomers from a maltooligosaccharide primer to produce α(1→4)-glucan—i.e., amylose. In this study, by exploiting the weak specificity for the substrate recognition of a thermostable α-glucan phosphorylase (from Aquifex aeolicus VF5), we investigated the enzymatic copolymerization of 2-deoxy-α-d-glucose 1-phosphate (dGlc-1-P), which was produced in situ from d-glucal, with Glc-1-P to obtain non-natural heteropolysaccharides composed of α(1→4)-linked dGlc/Glc units—i.e., partially 2-deoxygenated amylose. The reactions were carried out at different monomer feed ratios using a maltotriose primer at 40 °C for 24 h. The products were precipitated from the reaction medium, isolated by centrifugation, and subjected to 1H NMR spectroscopic and powder X-ray diffraction measurements to evaluate their chemical and crystalline structures, respectively. Owing to its amorphous nature, the partially 2-deoxygenated amylose with adapted unit ratios formed a film when subjected to a casting method.

Dual-enzyme co-immobilization for the one-pot production of glucose 6-phosphate from maltodextrin

Abstract D-tagatose (D-tag) is a natural yet rare hexose that can be used as a functional sweetener owing to its hypoglycemic and anticaries properties. A four-step enzymatic cascade reaction was performed to synthesize d -tagatose from maltodextrin. In this study, we constructed a catalytic cascade system by co-immobilizing α-glucan phosphorylase (αGP) and phosphoglucomutase (PGM) on Duolite A568 exchange resin to produce glucose 6-phosphate (G6P), a precursor of d -tagatose. Our results showed that the thermal stability of co-immobilized αGP&PGM was enhanced comparing to free enzymes. Co-immobilized αGP&PGM retained 63.4% activity after incubating at 75 °C for 36 h, whereas free enzymes retained only 12% activity. Moreover, the relative activity of co-immobilized αGP&PGM was consistently maintained above 78.5% after eight cycles of reuse, and its storage stability was approximately eleven times higher than that of the free enzymes after storage at 4 °C for 60 days. Notably, we successfully co-immobilized dual enzymes on the resin using poly (ethylene glycol) diglycidyl ether, which was found to be more effective than glutaraldehyde. The results suggested that co-immobilized αGP&PGM could act as a promising catalyst for the one-pot production of G6P and be used in the starting pathway for the further production of d -tagatose.

Efficient Multi-Enzymes Immobilized on Porous Microspheres for Producing Inositol From Starch.

In vitro synthetic enzymatic biosystem is considered to be the next generation of biomanufacturing platform. This biosystem contains multiple enzymes for the implementation of complicated biotransformatiom. However, the hard-to-reuse and instability of multiple enzymes limit the utilization of this biosystem in industrial process. Multi-enzyme immobilization might be a feasible alternative to address these problems. Herein, porous microspheres are used as carriers to co-immobilize multiple enzymes for producing inositol from starch. At first, all the enzymes (i.e., α-glucan phosphorylase aGP, phosphoglucose mutase PGM, inositol 1-phosphate synthase IPS, and inositol monophosphatase IMP) for converting starch to inositol were immobilized on porous microspheres individually to check the effect of immobilization, then all the enzymes are co-immobilized on porous microspheres. Compared to reaction system containing all the individual immobilized enzymes, the reaction system containing the co-immobilized enzymes exhibit ∼3.5 fold of reaction rate on producing inositol from starch. This reaction rate is comparable to that by free enzyme mixture. And the co-immobilized multi-enzyme system show higher thermal stability and recovery stability than free enzyme mixture. After 7 batches, the immobilized enzymes retain 45.6% relative yield, while the free enzyme mixture only retain 13.3% relative yield after 3 batches. Co-immobilization of multiple enzymes on porous microspheres for biomanufacturing would shed light on the application of in vitro synthetic enzymatic biosystem in industrial scale.

The plant-type phosphoenolpyruvate carboxylase Gmppc2 is developmentally induced in immature soy seeds at the late maturation stage: a potential protein biomarker for seed chemical composition.

Phosphoenolpyruvate carboxylase (PEPC) is a carbon-fixing enzyme with critical roles in seed development. Previously we observed a positive correlation between PEPC activity and protein content in mature seeds among soybean cultivars and varietal differences of PEPC activity in immature seeds, which is concordant with seed protein accumulation. Here, we report a PEPC isoform (Gmppc2) which is preferentially expressed in immature soybean seeds at the late maturation stage. Gmppc2 was co-expressed with enzyme genes involved in starch degradation: α-amylase, hexokinase, and α-glucan phosphorylase. Gmppc2 was developmentally induced in the external seed coats, internal seed coats, hypocotyls, and cotyledons at the late maturation stage. The expression of Gmppc2 protein was negatively regulated by the application of a nitrogen fertilizer, which suppressed nodule formation. These results imply that Gmppc2 is involved in the metabolism of nitrogen originated from nodules into seeds, and Gmppc2 might be applicable as a biomarker of seed protein content.Abbreviations: PEP: phosphoenolpyruvate; PEPC: phosphoenolpyruvate carboxylase; RNA-Seq: RNA sequencing; PCA: principal component analysis; SE: standard error.