融合淀粉酶AmyP-Clo对大米生淀粉的高效降解

【目的】获得能高效降解大米生淀粉的α-淀粉酶。【方法】将来源Clostridium butyricum T-7的α-淀粉酶的淀粉结合结构域与能快速偏好性降解大米生淀粉的α-淀粉酶Amy P的催化结构域融合重组表达。【结果】融合蛋白Amy P-Clo保留了野生型Amy P催化优势的基础上,对大米生淀粉的比活为(373.9±8.4)U/mg,4 h内对5%大米生淀粉的最终降解率为(42.7±1.1)%,对大米生淀粉的最高结合率为(71.1±1.6)%,这些数据相比Amy P分别提高了3.1、2.8和1.3倍。【结论】融合蛋白Amy P-Clo能高效降解生大米淀粉,是一个具有优良应用价值的酶。     

马铃薯淀粉合成与降解研究进展

马铃薯是我国第四大粮食作物,其总产量占全国粮食产量的20%。马铃薯是C3作物,其淀粉含量相对较低,这与其淀粉合成和降解过程有关。与马铃薯淀粉合成的相关酶包括腺苷二磷酸葡萄糖焦磷酸化酶(ADP-glucose pyrophosphorylase,AGPase)、颗粒结合性淀粉合成酶(Granule-bound starch synthase,GBSS)、可溶性淀粉合成酶(Soluble starch synthase,SSS)、淀粉分支酶(Starch branching enzyme,SBE)和淀粉去分支酶(Starch debranching enzyme,DBE)。α-淀粉酶(α-amylase,Amy)、β-淀粉酶(β-amylase,BAM)、葡聚糖水双激酶(Glucan water dikinase,GWD)、磷酸葡聚糖水双激酶(Phosphoglucan water dikinase,PWD)和酸性转化酶(Acid invertase,AI)、中/碱性转化酶(Neutral/alkaline inverstase,NI)则与马铃薯淀粉降解有关。马铃薯淀粉合成与降解基因克隆对马铃薯品种遗传改良具有重要意义,目前部分克隆的马铃薯淀粉合成与降解基因已在增加马铃薯淀粉含量、淀粉改性和改变淀粉组分方面得到广泛应用。总结了马铃薯淀粉合成与降解酶基因的研究进展,并提出今后研究建议,旨为深入开展马铃薯淀粉改良工程研究提供参考。     

猴头菌固体发酵降解玉米淀粉的初步研究

猴头菌在玉米粉培养基上进行固体发酵时能够产生活性较强的α－淀粉酶降解玉米淀粉。添加5％～15％黄豆粉和发酵时间从15d延长到25d能显著提高淀粉酶活力和淀粉降解率。在添加15％黄豆粉,加水40％,25℃发酵25d的玉米粉发酵产物中,淀粉降解率达到65．20％;蛋白质含量达到16．83％;蛋白质中赖氨酸含量由35．96mg／g提高到56．12mg／g,色氨酸含量由9．05mg／g提高到13．25mg／g,从而明显提高了玉米粉的营养价值。     

β-淀粉酶对磷酸化酶的“抑制作用”

磷酸化酶和β-淀粉酶是在淀粉合成和分解过程中起主要作用的酶,在植物体中往往同时存在,因此研究这两个酶相互之间的关系是很有意义的。Porter 曾报告不同来源的β-淀粉酶在溶液中对磷酸化酶有抑制作用,她认为此抑制作用的机制主要是β-淀粉酶直接影响磷酸化酶的活动中心。但是我们都知道,β-淀粉酶是分解淀粉的,而磷酸化酶的形成淀粉的作用需要先有淀粉作为引子(primer),当引子量低于一定浓     

一株降解生木薯淀粉的曲霉菌株的筛选、鉴定及其淀粉降解特性

本研究利用以生木薯淀粉为唯一碳源的筛选培养基,从腐烂木薯渣中分离筛选出一株可以降解生木薯淀粉的真菌菌株RSDF-7.根据RSDF-7形态和18S rDNA与28S rDNA之间的内转录间隔区(internal transcribed spacer,ITS)序列分析的结果,初步认定该菌株为曲霉属.菌株RSDF-7的粗酶液对多种不同的生淀粉底物均有水解效果;在以大米和玉米淀粉为底物时,其生淀粉分解活力比较高,分别为42%和40%.菌株RSDF-7的粗酶液具有良好的低pH稳定性,对生木薯淀粉的最适作用温度为50℃,最适作用pH为4.5.在30 min的吸附后,RSDF-7的粗酶液对生木薯淀粉的吸附力高达60%.使用HPLC对粗酶液的酶解产物进行检测,结果发现酶解产物中仅存在葡萄糖,表明菌株RSDF-7所产的生淀粉降解酶主要为糖化酶.扫描电镜观察结果发现,经RSDF-7粗酶液酶解后的生木薯粉颗粒破裂,形成空洞,说明RSDF-7粗酶液对生淀粉有较强的水解作用.可以预见,经纯化后的曲霉菌株RSDF-7生淀粉酶将来可以用于基于酶解的木薯淀粉转化.     

大豆叶片淀粉酶的特性

大豆叶片的α—淀粉酶对SH—反应剂不敏感,在pH3.6时失活Ca~(2 )使它的耐热性提高至70℃.β—淀粉酶对SH—反应剂敏感,耐酸至pH 2.3,耐热至60℃. 随着叶龄的增长α—淀粉酶的同工酶数目增加,此酶在一般情况下不需要添加Ca~(2 ),EDTA对它起不可逆的失活作用,β—极限糊精及Ca~(2 )可排除EDTA对酶的影响。此酶可能是Ca—金属酶,Ca~(2 )与酶结合紧密,它位于酶的活性中心或附近。 在体外,大豆叶片α—淀粉酶可被自身的淀粉粒吸附,但容易被 pH5的醋酸缓冲液洗脱。     

四种羊肚菌在固体发酵条件下的菌丝生物量和降解淀粉作用

本文对4种羊肚菌在固体发酵条件下的菌丝生物量和降解淀粉作用进行了研究,结果表明：羊肚菌(Morchella esculenta)、尖顶羊肚菌(M conica)、黑脉羊肚菌(M angusticeps)和皱柄羊肚菌(M. crassipes)在玉米粉培养基或马铃薯粉培养基上进行固体发酵时,菌丝生物量之间无显著差异;但a-淀粉酶活力、淀粉降解率差异显著。4种羊肚菌中,尖顶羊肚菌的降解淀粉能力最强。在培养基中添加Ca²⁺和氮源以及将发酵时间从15天延长到25天均能显著提高羊肚菌的菌丝生物量、a-淀粉酶活力和淀粉降解率。在添加10％黄豆粉、0.1％Ca(Cl)₂25℃发酵25天的玉米粉和马铃薯粉的发酵产物中,尖顶羊肚菌对玉米淀粉的降解率可达到74.2％,对马铃薯淀粉的降解率可达到79.8％。     

海洋环境来源的淀粉酶AmyP对生玉米 淀粉的降解特性

来自海洋宏基因组文库的 α-淀粉酶（AmyP）属于最新建立的糖苷水解酶亚家族GH13₃7。AmyP 是一个生淀粉降解酶,能有效降解玉米生淀粉。在最适反应条件 pH 7.5和 40 °C 下,生玉米淀粉的比活达到 39.6 ± 1.4 U/mg。酶解反应动力学显示 AmyP 可以非常快速的降解生玉米淀粉。对 1%的生玉米淀粉仅需要 30 min;4%和 8%的生玉米淀粉只需 3 h。DTT 可以显著提高 AmyP 对生玉米淀粉的降解活性,1% DTT 促使活性增加 1倍。根据电镜观察和产物分析,认为 AmyP 是以内腐蚀的模式降解生玉米淀粉颗粒,释放出葡萄糖、麦芽糖和麦芽三糖作为终产物。     

双酶法水解辐照改性马铃薯淀粉动力学研究

为了解辐照改性马铃薯淀粉的酶解特性,用α-淀粉酶和糖化酶同时作用于马铃薯原淀粉和经400 kGy剂量辐照处理后淀粉,考察了pH值、酶解温度、α-淀粉酶用量、糖化酶用量对反应速率的影响.以米氏方程为基础,用Lineweaver-Burk法求解动力学参数.结果表明,辐照后马铃薯淀粉的酶解反应速率明显高于马铃薯原淀粉.在单一水解体系中,α-淀粉酶和糖化酶对辐照前后马铃薯淀粉的降解都遵循Michaelis-Menten方程,α-淀粉酶的Km分别为11.343 mg· mL-1和9.386 mg· mL-1,Vmax分别为0.406 mg（mL·min）-1和1.079 mg（mL·min）-1;糖化酶的Km分别为10.307 mg· mL-1和8.905 mg·mL-1,Vmax分别为0.338 mg（mL·min）-1和0.821mg（mL·min）-1;水解产物葡萄糖对反应体系具有竞争性抑制剂的作用,其抑制常数Ki分别为1.298 mg·mL-1和0.934 mg·mL-1.研究结果表明辐照有效提高了马铃薯淀粉的酶解反应活性.     

光在黄化玉米叶片的苹果酸酶形成中的刺激作用

光刺激能使玉米黄化叶片的苹果酸酶活性提高,并随光强及照光时间的递增而加强。在照光条件下,抑制剂环已酰亚胺明显地抑制苹果酸酶活性的增加,同时,也抑制蛋白质合成以及叶绿素含量的增加。DCMU对酶的活性没有影响,表明在光下苹果酸酶活性的提高与光合电子传递无关。同位素放射自显影的结果:光刺激~3H-甘氨酸参入玉米黄化转绿叶片的苹果酸酶蛋白中。黄化叶片中的苹果酸酶在转绿过程中催化能力的提高是由于酶蛋白的重新合成。     

谷物籽粒淀粉研究进展

谷物籽粒是人类最基本的粮食,其胚乳淀粉与品质有密切的关系.本文就谷物胚乳淀粉的合成积累过程、谷物淀粉合成酶的作用与特性、淀粉特性与品质的关系以及基因工程在改良作物淀粉品质方面的研究进展进行了综述,并对谷物籽粒淀粉进一步的研究提出了展望,为谷物淀粉品质育种提供参考.     

Starch granule initiation in Arabidopsis thaliana chloroplasts

The initiation of starch granule formation and the mechanism controlling the number of granules per plastid have been some of the most elusive aspects of starch metabolism. This review covers the advances made in the study of these processes. The analyses presented herein depict a scenario in which starch synthase isoform 4 (SS4) provides the elongating activity necessary for the initiation of starch granule formation. However, this protein does not act alone; other polypeptides are required for the initiation of an appropriate number of starch granules per chloroplast. The functions of this group of polypeptides include providing suitable substrates (maltooligosaccharides) to SS4, the localization of the starch initiation machinery to the thylakoid membranes, and facilitating the correct folding of SS4. The number of starch granules per chloroplast is tightly regulated and depends on the developmental stage of the leaves and their metabolic status. Plastidial phosphorylase (PHS1) and other enzymes play an essential role in this process since they are necessary for the synthesis of the substrates used by the initiation machinery. The mechanism of starch granule formation initiation in Arabidopsis seems to be generalizable to other plants and also to the synthesis of long-term storage starch. The latter, however, shows specific features due to the presence of more isoforms, the absence of constantly recurring starch synthesis and degradation, and the metabolic characteristics of the storage sink organs.     

禾谷类作物淀粉合酶

淀粉合酶是禾谷类作物淀粉合成所必需的一类酶.根据淀粉合酶家族成员的氨基酸序列的相似性,分别介绍了一个颗粒性淀粉合酶亚家族和四个可溶淀粉合酶亚家族的组成、基因结构和表达特点,并从转录、转录后和翻译后水平上对这些基因的表达调控做了概述.     

Physicochemical properties of endosperm and pericarp starches during maize development

Endosperm starch and pericarp starch were isolated from maize (B73) kernels at different developmental stages. Starch granules, with small size (2–4 μm diameter), were first observed in the endosperm on 5 days after pollination (DAP). The size of endosperm-starch granules remained similar until 12DAP, but the number increased extensively. A substantial increase in granule size was observed from 14DAP (diameter 4–7 μm) to 30DAP (diameter10–23 μm). The size of starch granules on 30DAP is similar to that of the mature and dried endosperm-starch granules harvested on 45DAP. The starch content of the endosperm was little before 12DAP (less than 2%) and increased rapidly from 10.7% on 14DAP to 88.9% on 30DAP. The amylose content of the endosperm starch increased from 9.2% on 14DAP to 24.2% on 30DAP and 24.4% on 45DAP (mature and dried). The average amylopectin branch chain-length of the endosperm amylopectin increased from DP23.6 on 10DAP to DP26.9 on14DAP and then decreased to DP25.4 on 30DAP and DP24.9 on 45DAP. The onset gelatinization temperature of the endosperm starch increased from 61.3 °C on 8DAP to 69.0 °C on 14DAP and then decreased to 62.8 °C on 45DAP. The results indicated that the structure of endosperm starch was not synthesized consistently through the maturation of kernel. The pericarp starch, however, showed similar granule size, starch content, amylose content, amylopectin structure and thermal properties at different developmental stages of the kernel.     

Improved cold starch hydrolysis with urea addition and heat treatment at subgelatinization temperature

We explore how the presence of urea can influence the kinetics of amylolysis, with a long-term objective of developing practical and energy efficient bioconversion protocols. In this study, triticale and corn starches were hydrolyzed by a granular starch hydrolyzing enzyme with or without addition of urea and a pre-heating treatment at subgelatinization temperature. Differential scanning calorimetry showed that the gelatinization parameters of triticale and corn starches were negatively correlated with the urea concentration in the starch suspension. Addition of urea did not significantly affect starch amylolysis by the granular starch hydrolyzing enzyme at 30 °C. However when pre-heating at a higher yet sub-gelatinization temperature (50 °C for triticale and 61 °C for corn, 5 °C below the onset of starch gelatinization) for 30 min, the presence of urea greatly facilitated the amylolysis of both tricticale and corn starches. Scanning electron microcopy revealed starch granule mophological changes to a porous structure in residual starch granules/fragments rich in resistant starch. This means that the amylolysis pattern in the presence of urea was fundamentally changed, and urea disrupts starch hydrogen bonds effectively with heating treatment at a sub-gelatinization temperature. This treatment combination increased both starch hydrolysis rate and extent. Since extra energy was not necessary to gelatinize starch, this method may benefit starch and bio-enthanol industries to reduce the costs of starch hydrolysis.     

Effects of protein on crosslinking of normal maize, waxy maize, and potato starches

Channels of maize starch granules are lined with proteins and phospholipids. Therefore, when they are treated with reagents that react at or near the surfaces of channels, three types of crosslinks could be produced: protein–protein, protein–starch, starch–starch. To determine which of these may be occurring and the effect(s) of channel proteins (and their removal) on crosslinking, normal and waxy maize starches were treated with a proteinase (thermolysin, which is known to remove protein from channels) before and after crosslinking, and the properties of the products were compared to those of a control (crosslinking without proteinase treatment). After establishing that treatment of starch with thermolysin alone had no effect on the RVA trace, three reaction sequences were used: crosslinking alone (CL), proteinase treatment before crosslinking (Enz-CL), proteinase treatment after crosslinking (CL-Enz). Two crosslinking reagents were used: phosphoryl chloride (POCl₃), which is known to react at or near channel surfaces; STMP, which is believed to react throughout the granule matrix. Three concentrations of POCl₃ (based on the weight of starch) were used. For both normal maize starch (NMS) and waxy maize starch (WMS) reacted with POCl₃, the trends were generally the same, with apparent relative degrees of crosslinking indicated to be CL-Enz = CL > Enz-CL, but the effects were greater with NMS and there were differences when different concentrations of reagent were used. The basic trends were the same when potato starch was used in the same experiments. Crosslinking with STMP was done both in the presence and the absence of sodium sulfate (SS). Both with and without SS and with both NMS and WMS, the order of indicated crosslinking was generally the same as found after reaction with POCl₃, with the indicated swelling inhibition being greater when SS was present in the reaction mixture. Examination of the maize starches with a protein stain indicated that channel protein was removed by treatment with thermolysin when the proteinase treatment occurred before crosslinking with either POCl₃ or STMP, but only incompletely or not at all if the treatment with the proteinase occurred after crosslinking. Because the crosslinking reactions were less effective when the protein was removed, the results are tentatively interpreted as indicating that they involved protein molecules, although there may not be a direct relationship.     

Potato powders prepared by successive cooking-process depending on resistant starch content affect the intestinal fermentation in rats

The effects of resistant starch (RS) in dry potato powders prepared by various processes on intestinal fermentation in rats were assessed. Rats were fed raw potato powder (RP), blanched potato powder (BP), steamed potato powder (SP), or drum-dried potato powder (DP) for 4 weeks. The cecal RS content was significantly higher in the RP group than in the control diet (CN) group and other dry potato powder groups. Cecum pH was significantly lower in the RP group compared to the CN group, and was also significantly lower than that in the SP, BP, and DP groups. Lactic acid bacteria levels in the RP group were significantly higher than those in the CN group, and levels in the SP group also increased relative to the control group. Lactobacillus levels in the RP group were higher than in the CN and other dry potato powder groups. Cecal short-chain fatty acid (SCFA) concentrations in the RP group followed by the SP group exhibited significantly higher levels relative to the control levels. Dry potato powders containing RS produced during the cooking process may represent a useful food material that increases intestinal concentrations of SCFA and enhances the growth of certain lactic acid bacteria.     

Functional properties of pregelatinized and cross-linked cassava starch obtained by extrusion with sodium trimetaphosphate

Cassava starch was cross-linked with sodium trimetaphosphate (STMP) on a Cerealtec single-screw extruder at different extrusion temperatures and concentrations of STMP and NaOH. The effect of variables on functional properties of the products was analysed by the response–surface methodology. The degree of substitution (DS) was influenced by NaOH and phosphorus level, and increased when their concentration increased. Extrusion temperature affected water absorption, cold viscosity and gel characteristics. The introduction of phosphate groups by cross-linking, with maximum DS of 1.5×10⁻⁴, increased the gel strength, water absorption index, resistance to high temperature and shear, and decreased gel cohesiveness, starch clarity and water solubility index. The products had different DS and degree of gelatinization and thus can be applied in several kinds of foods.     

A genetic strategy generating wheat with very high amylose content

Resistant starch (RS), a type of dietary fibre, plays an important role in human health; however, the content of RS in most modern processed starchy foods is low. Cereal starch, when structurally manipulated through a modified starch biosynthetic pathway to greatly increase the amylose content, could be an important food source of RS. Transgenic studies have previously revealed the requirement of simultaneous down‐regulation of two starch branching enzyme (SBE) II isoforms both located on the long arm of chromosome 2, namely SBEIIa and SBEIIb, to elevate the amylose content in wheat from ~25% to ~75%. The current study revealed close proximity of genes encoding SBEIIa and SBEIIb isoforms in wheat with a genetic distance of 0.5 cM on chromosome 2B. A series of deletion and single nucleotide polymorphism (SNP) loss of function alleles in SBEIIa, SBEIIb or both was isolated from two different wheat populations. A breeding strategy to combine deletions and SNPs generated wheat genotypes with altered expression levels of SBEIIa and SBEIIb, elevating the amylose content to an unprecedented ~85%, with a marked concomitant increase in RS content. Biochemical assays were used to confirm the complete absence in the grain of expression of SBEIIa from all three genomes in combination with the absence of SBEIIb from one of the genomes.