Comparative Analysis of Primary and Secondary Structure for Pea Isoamylase Isoforms Predicts Different Catalytic Properties against Glucan Substrates

Screening of pea cDNA library has resulted in isolation of three different genes that encode different isoforms of isoamylase. From analysis and comparison with other isoamylase genes available from public databases and also from cDNA clones encoding isoforms of potato isoamylase, there are likely to be three isoforms of isoamylase in most (all) angiosperm genomes although so far only few plant species have their cDNA or genomic DNA characterised for these genes. Analysis of the primary structure of the isoforms of isoamylase from pea showed that all of them possess characteristics to bind glucan substrates. However, one of the isoform, Psisa2, most likely does not have catalytic activity. Since the analysis of this isoform concluded that it retained the characteristics to bind to glucans, Psisa2 might play a non‐catalytic role during the formation or degradation of starch.     

异淀粉酶水解豌豆淀粉及其在玻纤浸润中的应用

该文研究了豌豆淀粉的异淀粉酶水解过程,并将其水解产物用作玻纤浸润剂。结果表明,异淀粉酶对豌豆淀粉的水解采用点蚀和劈裂两种方式进行。水解对淀粉颗粒聚集行为有影响,具体表现为粒度、X射线衍射强度和黏度的波动。当酶解温度为50℃,酶解时间30 h,所得酶解淀粉液表面张力可降至40 mN/m,与玻纤临界表面张力30 mN/m接近。同时,酶解破坏使更多的淀粉羟基被暴露出来,因此,水解产物可以在玻纤表面轻易铺展,并与玻纤表面牢固结合。直接以水解淀粉悬浊液为浸润剂,玻纤拉丝过程可顺利进行,且能在玻纤表面形成均匀的保护膜,所得68 tex玻纤断裂强力平均达到25.0 N。     

Determination of Optimum Conditions for Enzymatic Debranching of Cassava Starch and Synthesis of Resistant Starch Type III using Central Composite Rotatable Design

Cassava starch was debranched by treatment with isoamylase and pullulanase and the yield of resistant starch type III (RS III) optimized with respect to starch solids concentration (7.5‐15%, w/v), incubation time (8‐24 h) and enzyme concentration using central composite rotatable design. Higher concentrations of pullulanase (10‐35 U/g starch) compared to isoamylase (30–90 mU/g starch) were required to give a similar degree of starch hydrolysis within the experimental domain. A clear debranching end‐point was identifiable by following the reducing value, blue value and β‐hydrolysis limit of cassava starches debranched using isoamylase. It was difficult to define a debranching endpoint of pullulanase treatment by these parameters due to contaminating α‐D ‐(1→4) activity. The yield of RS III was significantly higher in isoamylolysates and increased steadily with increasing degree of hydrolysis to peak at 57.3%. Purification of the debranched material further increased the RS III yield to 64.1%. Prolonged (24 h) hydrolysis of cassava starch with high concentration of pullulanase (35 U/g) gave lower RS III contents in the purified (34.2%) and unpurified (36.2%) hydrolysates compared to 49.5 and 62.4%, respectively, at moderate pullulanase concentration (22.5 U/g) and incubation time (16 h).     

An SEC−MALLS Study of Molecular Features of Water‐soluble Amylopectin and Amylose of Tef [Eragrostis tef (Zucc.) Trotter] Starches

The molecular features of five tef starches along with those of commercial normal maize starch were investigated by size‐exclusion chromatography with multi‐angle laser light scattering‐differential refractive index detection (SEC/MALLS‐DRI) after solubilization in water by cooking in a household pressure cooker. The weight‐average molar mass ( ) and weight‐average root‐mean square radius of gyration (<R_g>_w) of the amylopectin (AP) of tef starches ranged from 10.1×10⁷ g/mol (156 nm) to 16.5×10⁷ g/mol (205 nm) with a mean of 13.9×10⁷ g/mol (186 nm). The AP of the tef starches was considerably smaller than that of maize starch ( = 19.6×10⁷ g/mol, <R_g>_w = 207 nm). These considerably smaller AP molecules in tef starches were most probably responsible for the low paste viscosity of tef starches as compared to maize starch. In most tef starches, the polydispersity index (PI) of the AP was broader than that of the AP of maize starch. The intermediate fraction (IN) 1.0−1.6, mean = 1.1) of most tef starches were similar to those of the IN of maize starch. The amylose (AM) (range 1.5×10⁶−3.0×10⁶ g/mol, mean = 2.2×10⁶ g/mol) and size (range 176−214 nm, mean = 191 nm) of most tef starches was also apparently similar to that of the maize starch ( = 2.3×10⁶ g/mol, <R_g>_w = 193 nm), but the polymer distribution was narrower. The AM−iodine complex of the tef starches had a λ_max range of 611−679 nm and the absorption shifted toward longer wavelengths by 8−14 nm as compared to the maize starch AM−iodine complex. The blue value (absorption at λ_max) for 1 mg/mL of tef AM had a range of 2.3−2.8 (mean = 2.5), whereas for the maize starch, the mean was 2.2. The branched nature of tef starches was also investigated by debranching with isoamylase and determination of chain lengths (DP_n) of the branches by size exclusion chromatography with refractive index detector (SEC‐RI). The AP in tef starches had a polymodal distribution with a periodicity similar to that of cereal starches. The branches had DP_n values of A = 11, B1 = 16, B2 = 46 (range 46−47), B3 = 70 (range 69−72) and B4 = 118 (range 113−123). The outer (A + B1) chains were shorter than those of maize starch AP with abundance (74%, w/w) only slightly less than that of the maize starch (75%, w/w). The slow rate of retrogradation, the slightly lower percent crystallinity, the lower gelatinization temperatures and the lower gelatinization enthalpy observed for tef starches (as compared to maize starch) are probably related to the shorter outer (A + B1) chain lengths of their amylopectin molecules, and may be the foundation of the comparably good keeping quality of tef injera, the main staple in the Ethiopian diet.     

短杆菌异淀粉酶产生菌选育及酶作用性质研究

研究和讨论了一株短杆菌异淀粉酶的筛选,诱变,产酶条件及部分酶学特性。结果是摇瓶发酵液中酶活单位最高可达520u/ml.酶作用最适pH可低至5.0,最适温度可高至55℃。该酶对多种不同的含α-1,6-葡萄糖苷键的底物皆具有良好的水解作用。水解产物的分子量分布情况类似于假单胞菌异淀粉酶。