Red Pullulan

Procedures for the quantitative extraction, activation, and assay of limit-dextrinase in cereal flours have been developed. Extraction and activation require incubation in buffer containing 20mm cysteine for at least 16 h or with 25mM dithiothreitol for 5 h. Activity is assayed with a soluble, dyed substrate (Red-Pullulan) or an insoluble, dyed, and cross-linked substrate (Azurine-CL-Pullulan) which is dispensed in tablet form (Limit-DextriZyme tablets).

Pullulanases are well‐known debranching enzymes hydrolyzing α‐1,6‐glycosidic linkages. To date, engineering of pullulanase is mainly focused on catalytic pocket or domain tailoring based on structure/sequence information. Saturation mutagenesis‐involved directed evolution is, however, limited by the low number of mutational sites compatible with combinatorial libraries of feasible size. Using Bacillus naganoensis pullulanase as a target protein, here we introduce the ‘evolutionary coupling saturation mutagenesis’ (ECSM) approach: residue pair covariances are calculated to identify residues for saturation mutagenesis, focusing directed evolution on residue pairs playing important roles in natural evolution. Evolutionary coupling (EC) analysis identified seven residue pairs as evolutionary mutational hotspots. Subsequent saturation mutagenesis yielded variants with enhanced catalytic activity. The functional pairs apparently represent distant sites affecting enzyme activity.

Type I pullulanases are enzymes that specifically hydrolyse α-1,6 linkages in polysaccharides. This study reports the analyses of a novel type I pullulanase (PulASK) from Anoxybacillus sp. SK3-4. Purified PulASK (molecular mass of 80 kDa) was stable at pH 5.0-6.0 and was most active at pH 6.0. The optimum temperature for PulASK was 60°C, and the enzyme was reasonably stable at this temperature. Pullulan was the preferred substrate for PulASK, with 89.90 % adsorbance efficiency (various other starches, 56.26–72.93 % efficiency). Similar to other type I pullulanases, maltotriose was formed on digestion of pullulan by PulASK. PulASK also reacted with β-limit dextrin, a sugar rich in short branches, and formed maltotriose, maltotetraose and maltopentaose. Nevertheless, PulASK was found to preferably debranch long branches at α-1,6 glycosidic bonds of starch, producing amylose, linear or branched oligosaccharides, but was nonreactive against short branches; thus, no reducing sugars were detected. This is surprising as all currently known type I pullulanases produce reducing sugars (predominantly maltotriose) on digesting starch. The closest homologue of PulASK (95 % identity) is a type I pullulanase from Anoxybacillus sp. LM14-2 (Pul-LM14-2), which is capable of forming reducing sugars from starch. With rational design, amino acids 362-370 of PulASK were replaced with the corresponding sequence of Pul-LM14-2. The mutant enzyme formed reducing sugars on digesting starch. Thus, we identified a novel motif involved in substrate specificity in type I pullulanases. Our characterization may pave the way for the industrial application of this unique enzyme.

An activity-based screening approach led to the identification of a novel glycoside hydrolase family-13 pullulanase gene (pul13A) from a psychrophilic bacterium. The recombinant enzyme exhibited a deduced peptide sequence of 1440 amino acid residues and was produced in a heterologous host in Escherichia coli. Purification from inclusion bodies was achieved by a six-step dialysis protocol enabling mild refolding of the urea-denaturated protein followed by affinity- and size-exclusion chromatography under native conditions. Pul13A is a type-I pullulanase, which is capable of hydrolysing α-1,6-glycosidic bonds in pullulan to produce maltotriose, while maltose and intermediate oligosaccharides are produced from soluble starch and amylopectin. The recombinant enzyme exhibited typical properties of cold-adapted proteins including low thermostability at elevated temperatures. It showed a temperature optimum at 35°C, while at 10°C residual activity (25%) remained. The optimal pH was in the range of 6.0-7.0, with Pul13A being stable at neutral and basic pH, but not in the acidic range. Catalytic activity was increased in the presence of divalent cations calcium and cobalt and both metal ions were also able to restore catalytic activity of EDTA-chelated enzyme samples. Pul13A represents the first type-I pullulanase from a psychrophile that has been produced in recombinant form. Moreover, its favourable enzymatic properties make this enzyme a potential candidate for industrial applications such as starch degradation for ethanol based biofuel production.

Expression of a limit-dextrinase (LD) type starch debranching enzyme (EC 3.2.1.41) was observed in developing wheat (Triticum aestivum L.) endosperm and germinating grains, indicating a role for the enzyme in both biosynthesis and degradation of starch. A full-length cDNA, TaLD1, encoding LD in wheat developing kernels was isolated and predicted to encode a 98.6 kDa mature protein active in amyloplasts. Isolated cDNA was expressed in Escherichia coli to produce a recombinant His-tagged LD, which mainly accumulated in inclusion bodies as an inactive enzyme. Extraction of His-tagged LD from the inclusion bodies followed by dialysis under thiol/disulphide redox conditions allowed partial refolding of the protein and detection of pullulanase specific activities by zymogram analysis and enzyme assays. Several active conformations were demonstrated by the recombinant TaLD1 and pullulanase activity could be modulated by redox conditions in vitro. The results suggest that cellular redox conditions may regulate the level of LD activity in wheat tissues.

Starch debranching enzymes (DBE) are required for mobilization of carbohydrate reserves and for the normal structural organization of storage glucan polymers. Two isoforms, the pullulanase-type DBEs and the isoamylase-type DBEs, are both highly conserved in plants. To address DBE functions in starch assembly and breakdown, this study characterized the biochemical activity of ZPU1, a pullulanase-type DBE that is the product of the maize Zpu1 gene. Assays showed directly that recombinant ZPU1 (ZPU1r) expressed in Escherichia coli functions as a pullulanase-type enzyme, and ¹H-NMR spectroscopy demonstrated that ZPU1r specifically hydrolyzes α-(1→6) branch linkages. Preferred substrates for ZPU1r hydrolytic activity were determined, as were pH, temperature, and thermal stability optima. Kinetic properties of ZPU1r with respect to two substrates, β-limit dextrin and pullulan, were determined. ZPU1 activity was increased by incubation with thioredoxin h, and native activity was decreased in mutants that accumulate soluble sugars, suggesting potential regulatory mechanisms.

A psychrophilic, aerobic bacterium designated A2i^T was isolated from marine sediment recovered from shallow waters surrounding Adelaide Island, Antarctica (67° 34' S, 68° 07' W). The organism exhibited xylanolytic and laminarinolytic activity and was halotolerant. Basic characterization showed that it was gram-negative, non-motile, yellow-pigmented (β, β-carotene-3,3'-diol) and positive for oxidase and catalase synthesis. Analysis of the 16S rDNA sequence suggests that the organism belongs to the Flexibacter-Cytophaga-Bacteroides phylum. On the basis of its 16S rDNA sequence, the bacterium is 96.8% similar to Flavobacterium columnare ATCC 43622 - its closest relation. The genomic DNA G+C content was 35 mol%. Growth on xylan occurs optimally at 15°C, though growth also occurs at 0°C, and the doubling times are 9.6 and 34.8 h, respectively. The maximum growth temperature on xylan is at 24°C. The bacterium is a neutrophile, growing across the pH range 5.6-8.4 and having an optimum at pH 7.5. Analysis of the 16S rDNA sequence, together with phenotypic characterization, suggests that the organism is a member of the genus Flavobacterium. DNA-DNA hybridization experiments have shown that it is a novel species; it is proposed, therefore, that the organism be designated as the type strain of Flavobacterium frigidarium sp. nov. (= ATCC 700810^T = NCIMB 13737^T).

We have isolated the gene encoding the neopullulanase enzyme from Bacillus polymyxa CECT 155. It consists of an open reading frame of 1545 bp that could code for a protein of 515 amino acids. This open reading frame was expressed in Bacillus subtilis and the corresponding transformants produced extracellular neopullulanase. The neopullulanase gene was also expressed in Saccharomyces cerevisiae placing it under the control of the yeast actin gene (ACT1) promoter. Clones containing the intact neopullulanase gene, including its own bacterial signal sequence, gave rise to the synthesis of active, but intracellular, enzyme by S.cerevisiae transformants. When sequences specifying the signal sequence and leader region of the yeast mating pheromone α-factor (MFα1) were fused upstream of the gene encoding the neopullulanase enzyme, the enzyme was secreted by S. cerevisiae. The secreted protein presented the same biochemical properties and the same apparent molecular mass as the Bacillus polymyxa original enzyme. The predicted amino acid sequence of the neopullulanase protein contained sequence motifs conserved among amylolytic enzymes. Northern blot analysis indicated that the transcription of the neopullulanase gene in B. polymyxa was induced by the presence of the substrate, pullulan, in the culture, and was repressed by glucose.

The activities of the two types of starch debranching enzymes, isoamylase and pullulanase, were greatly reduced in endosperms of allelic sugary-1 mutants of rice (Oryza sativa), with the decrease more pronounced for isoamylase than for pullulanase. However, the decrease in isoamylase activity was not related to the magnitude of the sugary phenotype (the proportion of the phytoglycogen region of the endosperm), as observed with pullulanase. In the moderately mutated line EM-5, the pullulanase activity was markedly lower in the phytoglycogen region than in the starch region, and isoamylase activity was extremely low or completely lost in the whole endosperm tissue. These results suggest that both debranching enzymes are involved in amylopectin biosynthesis in rice endosperm. We presume that isoamylase plays a predominant role in amylopectin synthesis, but pullulanase is also essential or can compensate for the role of isoamylase in the construction of the amylopectin multiple-cluster structure. It is highly possible that isoamylase was modified in some sugary-1 mutants such as EM-273 and EM-5, since it was present in significant and trace amounts, respectively, in these mutants but was apparently inactive. The results show that the Sugary-1 gene encodes the isoamylase gene of the rice genome.

Biochemically active wheat thioredoxin h has been overexpressed in the endosperm of transgenic barley grain. Two DNA constructs containing the wheat thioredoxin h gene (wtrxh) were used for transformation; each contained wtrxh fused to an endosperm-specific B₁-hordein promoter either with or without a signal peptide sequence for targeting to the protein body. Twenty-two stable, independently transformed regenerable lines were obtained by selecting with the herbicide bialaphos to test for the presence of the barherbicide resistance gene on a cotransformed plasmid; all were positive for this gene. The presence of wtrxh was confirmed in 20 lines by PCR analysis, and the identity and level of expression of wheat thioredoxin h was assessed by immunoblots. Although levels varied among the different transgenic events, wheat thioredoxin h was consistently highly expressed (up to 30-fold) in the transgenic grain. Transgenic lines transformed with the B₁-hordein promoter with a signal peptide sequence produced a higher level of wheat thioredoxin h on average than those without a signal sequence. The overexpression of thioredoxin h in the endosperm of germinated grain effected up to a 4-fold increase in the activity of the starch debranching enzyme, pullulanase (limit dextrinase), the enzyme that specifically cleaves α-1,6 linkages in starch. These results raise the question of how thioredoxin h enhances the activity of pullulanase because it was found that the inhibitor had become inactive before the enzyme showed appreciable activity.