Synthesis of branched cyclomalto-oligosaccharides using Pseudomonas isoamylase

Branched cyclomalto-oligosaccharides (cyclodextrins) were synthesised from cyclomalto-oligosaccharides and maltose or maltotriose through the reverse action of Pseudomonas isoamylase. The reaction rate was greater with maltotriose than with maltose, and with increasing size of the cyclomalto-oligosaccharide (cG₆ < cG₇ < cG₈). Maltotriose is effective as both a side-chain donor and acceptor, and three isomers of 6-O-α-maltotriosylmaltotriose (branched G₆) were formed through mutual condensation, but maltose was effective only as a side-chain donor. Each branched cyclomalto-oligosaccharide and G₆ was purified by liquid chromatography, and their structures were determined by chemical, enzymic, and ¹³C-n.m.r. spectroscopic analyses.     

Production enhancement of Pseudomonas amyloderamosa isoamylase

Isoamylase production in batchwise and fed-batch cultures of Pseudomonas amyloderomosa (strain WU7211-2) was investigated. By feeding maltose in a mode motivated by its structure gene (iam), the final isoamylase activity of 5100 U/ml was achieved, as compared to 4100 U/ml in batch cultivation and 3800 U/ml in shaken flasks. The enhancement may be due to the fact that the production of isoamylase can be induced effectively by the maltose only if the glucose concentration is maintained below the inhibitory level. Also, cultivations based on 500-ml shaken flasks, performed with different amount of proteimax HE90, showed that higher amounts of proteimax HE90 resulted in an increased value of pH that had an adverse effect on isoamylase yield.The authors wish to thank the Food Industry Research and Development Institute (FIRDI), Taiwan, R.O.C for the financial support (Cooperative Agreement 94M904-2B). Appreciation is also extended to Drs. C.C. Liao, W.S. Chu and L.L. Lin of FIRDI for their valuable discussions.     

Preliminary X-ray studies on Pseudomonas isoamylase

Single crystals of Pseudomonas isoamylase (M_r 95,000) belong to space group P2₁2₁2₁ with unit cell dimensions of $\text{a = 137.9}\text{A}\text{?}\text{, b = 52.9}\text{A}\text{?}\text{, c = 151.2}\text{A}\text{?}$. A Guinier plot of the X-ray small-angle scattering of the protein solution gave the radius of gyration of the molecule as 27.5 Å.     

Expression of Pseudomonas amyloderamosa isoamylase gene in Saccharomyces cerevisiae

Isoamylase gene (iso) of Pseudomonas amyloderamosa was amplified by polymerase chain reaction and cloned into Saccharomyces cerevisiae vectors under the control of alcohol dehydrogenase gene and glyceraldehyde-3-phosphate dehydrogenase gene promoters. The signal sequence of iso gene was also replaced with that of Schwanniomyces occidentalis -amylase gene. The extracellular isoamylase activity of transformed Sacc. cerevisiae could reach 86 U ml–1 after a 4-days cultivation. © Rapid Science Ltd. 1998     

Mutants of Arabidopsis lacking a chloroplastic isoamylase accumulate phytoglycogen and an abnormal form of amylopectin

Mutant lines defective for each of the four starch debranching enzyme (DBE) genes (AtISA1, AtISA2, AtISA3, and AtPU1) detected in the nuclear genome of Arabidopsis (Arabidopsis thaliana) were produced and analyzed. Our results indicate that both AtISA1 and AtISA2 are required for the production of a functional isoamylase-type of DBE named Iso1, the major isoamylase activity found in leaves. The absence of Iso1 leads to an 80% decrease in the starch content in both lines and to the accumulation of water-soluble polysaccharides whose structure is similar to glycogen. In addition, the residual amylopectin structure in the corresponding mutant lines displays a strong modification when compared to the wild type, suggesting a direct, rather than an indirect, function of Iso1 during the synthesis of amylopectin. Mutant lines carrying a defect in AtISA3 display a strong starch-excess phenotype at the end of both the light and the dark phases accompanied by a small modification of the amylopectin structure. This result suggests that this isoamylase-type of DBE plays a major role during starch mobilization. The analysis of the Atpu1 single-mutant lines did not lead to a distinctive phenotype. However, Atisa2/Atpu1 double-mutant lines display a 92% decrease in starch content. This suggests that the function of pullulanase partly overlaps that of Iso1, although its implication remains negligible when Iso1 is present within the cell.     

Complementation of sugary-1 phenotype in rice endosperm with the wheat isoamylase1 gene supports a direct role for isoamylase1 in amylopectin biosynthesis

To examine the role of isoamylase1 (ISA1) in amylopectin biosynthesis in plants, a genomic DNA fragment from Aegilops tauschii was introduced into the ISA1-deficient rice (Oryza sativa) sugary-1 mutant line EM914, in which endosperm starch is completely replaced by phytoglycogen. A. tauschii is the D genome donor of wheat (Triticum aestivum), and the introduced fragment effectively included the gene for ISA1 for wheat (TaISA1) that was encoded on the D genome. In TaISA1-expressing rice endosperm, phytoglycogen synthesis was substantially replaced by starch synthesis, leaving only residual levels of phytoglycogen. The levels of residual phytoglycogen present were inversely proportional to the expression level of the TaISA1 protein, although the level of pullulanase that had been reduced in EM914 was restored to the same level as that in the wild type. Small but significant differences were found in the amylopectin chain-length distribution, gelatinization temperatures, and A-type x-ray diffraction patterns of the starches from lines expressing TaISA1 when compared with wild-type rice starch, although in the first two parameters, the effect was proportional to the expression level of TaISA. The impact of expression levels of ISA1 on starch structure and properties provides support for the view that ISA1 is directly involved in the synthesis of amylopectin.     

Production of isoamylase by Pseudomonas amyloderamosa mutant strain JD210

Nutritional requirements for the production of isoamylase by Pseudomonas amyloderamosa mutant strain JD210 were investigated. The optimal initial pH for enzyme production in shake-flask cultivation was 5.0. Maltose and soybean protein hydrolyzate were found to be the best carbon source and nitrogen source, respectively. The enzyme production was drastically inhibited by Zn+2 and Cu+2. Other metal ions phosphates and surfactants exhibited no significant inhibitory or accelerating effect on enzyme production. According to auxanography and single omission experiments, proline and isoleucine were required for growth. The supplement of 0.1% proline increased enzyme production by around 30% compared with no addition.     

Investigation on the immobilization of Pseudomonas isoamylase onto polysaccharide matrices

This study examined Pseudomonas isoamylase immobilized onto polysaccharide matrices, among which included agarose, cellulose, and raw corn starch. For chemical binding of polysaccharides activated with tosyl chloride, a high specific activity of 23144?U/g-starch was obtained as compared with matrices of cellulose and agarose with 3229?U/g-cellulose and 84?U/g-agarose, respectively. For raw corn starch, isoamylase desorption occurred when the immobilized enzyme by physical adsorption was subjected to 0.05?M acetate buffer with pH?5.2 at 40?°C; this is despite the considerable affinity between the enzyme and the matrix. In contrast, no detectable activity leached from the matrix for chemical binding, regardless of whether maltose, i.e. an affinity species to isoamylase, was added. For immobilized starch-isoamylase, its optimal activity performance was obtained in broader pH?ranges of 3.5–5.5 and 5?°C higher than those of the free enzymes. More specifically, the free enzyme's activity markedly decreased within five hours while the immobilized starch-isoamylase exhibited a fairly stable behavior over a three day incubation period at 40?°C. After 175 days of storage at 4?°C, the residues of relative activity of 75% and 45% were obtained with respect to immobilized and free isoamylases, respectively.     

Antisense inhibition of isoamylase alters the structure of amylopectin and the physicochemical properties of starch in rice endosperm

This is the first report on regulation of the isoamylase1 gene to modify the structure of amylopectin and properties of starch by using antisense technology in plants. The reduction of isoamylase1 protein by about 94% in rice endosperm changed amylopectin into a water-insoluble modified amylopectin and a water-soluble polyglucan (WSP). As compared with wild-type amylopectin, the modified amylopectin had more short chains with a degree of polymerization of 5-12, while their molecular sizes were similar. The WSP, which structurally resembled the phytoglycogen in isoamylase-deficient sugary-1 mutants, accounted for about 16% of the total alpha-polyglucans in antisense endosperm, and it was distributed throughout the whole endosperm unlike in sugary-1 mutant. The reduction of isoamylase activity markedly lowered the gelatinization temperature from 54 to 43 degrees C and the viscosity, and modified X-ray diffraction pattern and the granule morphology of the starch. The activity of pullulanase, the other type of starch debranching enzyme, in the antisense endosperm was similar to that in wild-type, whereas it is deficient in sugary-1 mutants. These results indicate that the isoamylase1 is essential for amylopectin biosynthesis in rice endosperm, and that alteration of the isoamylase activity is an effective means to modify the physicochemical properties and granular structure of starch.     

Molecular weight of the undegraded polypeptide chain of Pseudomonas amyloderamosa isoamylase

Crystalline isoamylase of Pseudomonas amyloderamosa was found to be contaminated with a trace of proteolytic enzyme. This contaminant digested the isoamylase under neutral or alkaline conditions, especially in the presence of sodium dodecyl sulfate (SDS). A reliable molecular weight of the enzyme was obtained by SDS-polyacrylamide gel electrophoresis and by gel filtration on Sepharose-6B in 6 M guanidine-hydrochloride after heat inactivation of the contaminant. The molecular weight of the undergraded polypeptide chain of the isoamylase was about 90 000. The lower molecular weight and the subunit structure of the enzyme reported previously are incorrect.     

Selection of antibiotic-resistant mutants with enhanced isoamylase activity in Pseudomonas amyloderamosa

Summary Isoamylase-hyperproducing strains of Pseudomonas amyloderamosa were bred by mutagenesis with UV light and N-methyl-N-nitro-N-nitrosoguanidine (NTG). The selection criterion for such strains was based on the formation of large turbid zones around the bacterial colonies in agar medium containing antibiotics and 1% waxy corn starch. Mutant WN6410 was obtained by treating P. amyloderamosa JD210 with five cycles of 1 × 10⁴ J UV light and one cycle of NTG. P. amyloderamosa WN6410 had 22-fold increase in isoamylase activity when compared to wild-type strain SB15 and the maximal enzyme activity, 5,100 U/ml, could be achieved within 48 h in 2.5 L fed-batch fermentation.     

Effect of pH on isoamylase production by Pseudomonas amyloderamosa WU 5315

The isoamylase activity of Pseudomonas amyloderamosa WU 5315 was stable over the pH range from 5.5 to 6.25 while only about 30% of the activity remained at pH 6.5. Low isoamylase activity (418 U ml^-1) was produced by the cells grown at high pH. Activity reached almost 3000 U ml^-1 when pH was kept below 6.0 during the fermentation. With 1% glucose plus 2% maltose instead of 3% maltose as carbon source, however, no pH control was required and the isoamylase activity of Ps. amyloderamosa WU 5315 increased to 3400 U ml^-1.     

Functional diversity of isoamylase oligomers: the ISA1 homo-oligomer is essential for amylopectin biosynthesis in rice endosperm

Rice (Oryza sativa) endosperm has two isoamylase (ISA) oligomers, ISA1 homo-oligomer and ISA1-ISA2 hetero-oligomer. To examine their contribution to starch synthesis, expression of the ISA1 or ISA2 gene was differently regulated in various transgenic plants. Although suppression of ISA2 gene expression caused the endosperm to have only the homo-oligomer, no significant effects were detected on the starch phenotypes. In contrast, ISA2 overexpression led to endosperm having only the hetero-oligomer, and starch synthesis in the endosperm was drastically impaired, both quantitatively and qualitatively, because the starch was devoid of typical starch features, such as thermal and x-ray diffraction properties, and water-soluble highly branched maltodextrins were accumulated. In the ISA2 overexpressed line, about 60% to 70% of the ISA1-ISA2 hetero-oligomer was bound to starch, while the ISA homo- and hetero-oligomers from the wild type were mostly present in the soluble form at the early milking stage of the endosperm. Detailed analysis of the relative amounts of homo- and hetero-oligomers in various lines also led us to the conclusion that the ISA1 homo-oligomer is essential, but not the ISA1-ISA2 oligomer, for starch production in rice endosperm. The relative amounts of ISA1 and ISA2 proteins were shown to determine the ratio of both oligomers and the stoichiometry of both ISAs in the hetero-oligomer. It was noted when compared with the homo-oligomer that all the hetero-oligomers from rice endosperm and leaf and potato (Solanum tuberosum) tuber were much more stable at 40°C. This study provides substantial data on the structural and functional diversity of ISA oligomers between plant tissues and species.     

Studies on carbohydrate-metabolising enzymes Part XXVII. A comparison of the action of yeast isoamylase and bacterial pullulanase on amylopectin

The action of purified yeast isoamylase on amylopectin, like that of bacterial pullulanase, results in the hydrolysis of the outermost inter-chain linkages with the liberation of linear maltosaccharides having an average degree of polymerisation of approximately 15 -glucose residues. This hydrolytic action distinguishes yeast isoamylase from yeast amylo-(1→6)-glucosidase, which acts by a combination of transferase and glucosidase activities. The products of enzyme action on amylopectin are discussed in relation to the probable molecular structure of the polysaccharide.     

Hydrolysis of amylopectin by amylolytic enzymes: structural analysis of the residual amylopectin population

Amylopectin fine structures were studied following limited hydrolysis of gelatinised waxy maize starch by amylases with a different level of inner chain attack (LICA). This was done by size exclusion chromatography as well as by debranching the (partially hydrolysed) amylopectin samples and studying the size distributions of the released chains. α-Amylases from Bacillus amyloliquefaciens and Aspergillus oryzae, with a relatively high LICA, drastically altered amylopectin chain length distribution and reduced the amylopectin molecular size (MS) significantly even at a low to moderate degree of hydrolysis (DH). Porcine pancreatic α-amylase (PPA), with a rather low LICA but a high multiple attack action on amylose, reduced the amylopectin MS much slower. Following hydrolysis by PPA to a DH of 10% and enzymic debranching of the amylopectin residue, several subpopulations of chains consisting of 2-12 glucose units were detected, indicating a multiple attack action on the amylopectin side chains. During the early stages of hydrolysis, the maltogenic Bacillus stearothermophilus α-amylase (BStA) preferentially hydrolysed the exterior chains of amylopectin. However, during the later phases, BStA also hydrolysed inner chains, presumably with a high multiple attack action. The present results clearly show that different enzymes can be used for (limited) conversion of amylopectin into structures differing in molecular weight and chain length distributions.     

Cloning and nucleotide sequence of the isoamylase gene from Pseudomonas amyloderamosa SB-15

The gene (iam) coding for isoamylase (glycogen 6-glucanohydrolase) of Pseudomonas amyloderamosa SB-15 was cloned. Its nucleotide sequence contained an open reading frame of 2313 nucleotides (771 amino acids) encoding a precursor of secreted isoamylase. The precursor contained a signal peptide of 26 amino acid residues at its amino terminus and three regions homologous with those conserved in alpha-amylases (1,4-alpha-D-glucan 4-glucanohydrolase) of species ranging from prokaryotes to eukaryotes. These homologous regions were also found in another debranching enzyme, pullulanase (pullulan 6-glucanohydrolase) from Klebsiella aerogenes. Sequences of the isoamylase also showed significant homology with those between positions 300 and the carboxyl terminus of pullulanase. The regions required for the specificity of isoamylase were discussed on the basis of a comparison of its amino acid sequence with those of alpha-amylases, cyclomaltodextrin glucanotransferases, and pullulanase.