环糊精葡萄糖基转移酶钙离子结合位点结构与功能分析

通过多重序列比对和晶体结构分析发现,钙离子结合位点CaⅠ和CaⅡ普遍存在于环糊精葡萄糖基转移酶(CGT酶)中,且两个位点处氨基酸残基具有较高的保守性,而钙离子结合位点CaⅢ仅存在于少数CGT酶中．此外,研究发现,钙离子结合位点可能与CGT酶的环化活力、热稳定性和产物特异性密切相关．     

环糊精糖基转移酶产物专一性改造：难题与挑战

生产环糊精所必需的环糊精糖基转移酶与其产物专一性进化研究已经成为当今的研究热点。这项研究中的进展不仅会使环糊精糖基转移酶的应用取得突破结果,还会对其它酶的改造提供帮助。目前,人们对这类酶的性质已经有了较为深入的认识,但还存在着一些尚未解决的问题,如酶产物专一性的决定因素尚未系统阐明等。通过对环糊精糖基转移酶各个方面,尤其是其产物专一性进化方面研究的回顾,指出并分析了研究中尚未解决的问题,并对将来这一研究领域的前景进行了展望。     

环糊精糖基转移酶产物专一性改造：难题与挑战

生产环糊精所必需的环糊精糖基转移酶与其产物专一性进化研究已经成为当今的研究热点。这项研究中的进展不仅会使环糊精糖基转移酶的应用取得突破结果,还会对其它酶的改造提供帮助。目前,人们对这类酶的性质已经有了较为深入的认识,但还存在着一些尚未解决的问题,如酶产物专一性的决定因素尚未系统阐明等。通过对环糊精糖基转移酶各个方面,尤其是其产物专一性进化方面研究的回顾,指出并分析了研究中尚未解决的问题,并对将来这一研究领域的前景进行了展望。     

Cyclomaltodextrin Glucanotransferase-Catalyzed Transglycosylation from Dextrin to Alkanol Maltosides

Maltosides of butanol, octanol, and lauryl alcohol were found for the first time to serve as substrates for cyclomaltodextrin glucanotransferase (CGTase), and glycosyl residue was transfered from dextrin to the substrate affording novel maltosides with 3–4 glucose units.     

魔芋葡甘露聚糖固定化环糊精葡基转移酶的研究

本文报道将魔芋葡甘露聚糖(简称KGM),经不溶性处理和一定的化学修饰活化作固定化载体,用共价键合法固定化环糊精葡基转移酶(简称CGTase)。其中,用表氯醇-已二胺-戊二醛修饰活化的KGM载体固定化CGTase效果最好,偶联蛋白质多在20mg/g载体以上,酶活500～900u/g载体之间,最高可超过52mg/g和1300u/g载体。固定化CGTase在pH5和pH10呈两个酶活峰值,最适温度60℃。以淀粉为底物批式连续反应,转化率均在90％以上。     

Characterization of an Archaeal Cyclodextrin Glucanotransferase with a Novel C-Terminal Domain

A gene encoding a cyclodextrin glucanotransferase (CGTase) from Thermococcus kodakaraensis KOD1 (CGT(Tk)) was identified and characterized. The gene (cgt(Tk)) encoded a protein of 713 amino acid residues harboring the four conserved regions found in all members of the alpha-amylase family. However, the C-terminal domain corresponding to domain E of previously known CGTases displayed a completely distinct primary structure. In order to elucidate the catalytic function of the gene product, the recombinant enzyme was purified by anion-exchange chromatography, and its enzymatic properties were investigated. The enzyme displayed significant starch-degrading activity (750 U/mg of protein) with an optimal temperature and pH of 80 degrees C and 5.5 to 6.0, respectively. The presence of Ca(2+) enhanced the enzyme activity and elevated the optimum temperature to 85 to 90 degrees C. With the addition of Ca(2+), the enzyme showed extreme thermostability, with almost no loss of enzymatic activity after 80 min at 85 degrees C, and a half-life of 20 min at 100 degrees C. CGT(Tk) could hydrolyze soluble starch and glycogen but failed to hydrolyze pullulan. Most importantly, although CGT(Tk) harbored a unique C-terminal domain, we found that the protein also exhibited significant CGTase activity, with beta-cyclodextrin as the main product. In order to identify the involvement, if any, of the C-terminal region in the CGTase activity, we analyzed a truncated protein (CGT(Tk)DeltaC) with 23 C-terminal amino acid residues deleted. CGT(Tk)DeltaC displayed similar properties in terms of starch-binding activity, substrate specificity, and thermostability, but unexpectedly showed higher starch-degrading activity than the parental CGT(Tk). In contrast, the cyclization activity of CGT(Tk)DeltaC was abolished. The results indicate that the presence of the structurally novel C-terminal domain is essential for CGT(Tk) to properly catalyze the cyclization reaction.     

Transglycosylation of Glycosyl Residues to Cyclic Tetrasaccharide by Bacillus stearothermophilus Cyclomaltodextrin Glucanotransferase Using Cyclomaltodextrin as the Glycosyl Donor

Cyclomaltodextrin glucanotransferase (EC 2.4.1.19, abbreviated as CGTase) derived from Bacillus stearothermophilus produced a series of transfer products from a mixture of cyclomaltohexaose and cyclic tetrasaccharide (cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→}, CTS). Of the transfer products, only two components, saccharides A and D, remained and accumulated after digestion with glucoamylase. The total combined yield of the saccharides reached 63.4% of total sugars, and enzymatic and instrumental analyses revealed the structures of both saccharides. Saccharide A was identified as4-mono-O-α-glucosyl-CTS, {→6)-[α-D-Glcp-(1→4)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→}, and sachharide D was 4,4′-di-O-α-glucosyl-CTS, {→6)-[α-D-Glcp-(1→4)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-[α-D-Glcp-(1→4)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→}. These structures led us to conclude that the glycosyltransfer catalyzed by CGTase was specific to the C4-OH of the 6-linked glucopyranosyl residues in CTS.     

植物体内蔗糖转运蛋白的功能和调控

蔗糖转运蛋白（sucrose transporter,SUT）负责蔗糖的跨膜运输,在韧皮部介导的源-库蔗糖运输和为库组织供应蔗糖的生理活动中起关键作用。本文介绍植物体内蔗糖转运蛋白基因家族、细胞定位与功能调节以及高等植物的蔗糖感受机制的研究进展。     

Hormonal Control of Sucrose Phosphate Synthase and Sucrose Synthase in Sugar Beet

We studied the effects of synthetic analogs of phytohormones (benzyladenine, IAA, and GA) on the activities of the enzymes catalyzing sucrose synthesis and metabolism, sucrose phosphate synthase (SPS, EC 2.4.1.14) and sucrose synthase (SS, EC 2.4.1.13), and on the content of chlorophyll and protein during the sugar-beet (Beta vulgaris L.) ontogeny. Plant spraying with phytohormonal preparations activated SPS in leaves; direct interaction between phytohormones and the enzyme also increased its activity. The degree of this activation differed during the ontogeny and in dependence on the compound used for treatment. Analogs of phytohormones maintained high protein level in leaves, retarded chlorophyll breakdown, and, thus, prolonged leaf functional activity during development. Phytohormonal preparations practically did not affect the SS activity both after plant treatment and at their direct interaction with the enzyme. It is supposed that the SS activity in sugar-beet roots is controlled by sucrose synthesized in leaves rather than by phytohormones. The effects of hormones on leaf metabolism were mainly manifested in growth activation.     

Engineering of Hydrolysis Reaction Specificity in the Transglycosylase Cyclodextrin Glycosyltransferase

Abstract

Cyclodextrin glycosyltransferase (CGTase) is a member of the α-amylase family, a large group of enzymes that act on α-glycosidic bonds in starch and related compounds. Over twenty different reaction and product specificities have been found in this family. Although three-dimensional structure elucidation and the biochemical characterization of site-directed mutants have yielded a detailed insight into the mechanism of bond cleavage, the variation in reaction and product specificity is far from understood. This article gives an overview of recent developments in the undersanding and engineering of transglycosylation and hydrolysis specificity in CGTase, which is one of the best-studied α-amylase family enzymes.     

环糊精葡萄糖基转移酶的性质、应用与固定化研究进展

本文总结了环糊精葡萄糖基转移酶的性质、应用与固定化研究的进展情况,引用文献４７篇。     

Anchorage of cyclodextrin glucanotransferase on the outer membrane of Escherichia coli

The gene encoding cyclodextrin glucanotransferase (CGTase) was successfully cloned from B. macerans by PCR. A recombinant plasmid pCS005 with a gene encoding the Lpp-OmpA-CGTase trifusion protein was constructed and transformed into E. coli for the surface display of CGTase. Results of immunoblotting analysis and protease accessibility on the fractionated cell membranes confirmed that the Lpp-OmpA-CGTase trifusion protein was successfully anchored on the outer membrane of E. coli. However, only 50% of the membrane-anchored trifusion proteins were displayed on the outer surface of E. coli with the remaining 50% un-translocated. The low efficiency of surface display is attributed to the large size of CGTase. Only a trace amount of CGTase activity was detected for both the whole cells and the cell debris fractions. Because the results of the protease accessibility study suggested that the trypsin-resistant conformation of CGTase was preserved in the membrane-anchored CGTase, we believe that the lack of enzyme activity is mainly due to the inaccessibility of the CGTase active site, near the N-terminus, for substrate molecules. It can be estimated that the critical size for surface display of protein in E. coli is approximately 70 kDa.     

小麦开花后旗叶中蔗糖合成与籽粒中蔗糖降解

在小麦开花后,旗叶中蔗糖磷酸合成酶（SPS）活性在开花后14d内一直维持较高水平,蔗糖合成酶（SS）的活性在开花后14-28d较高,蔗糖的含量与SPS活性呈显著正相关,籽粒中蔗糖合成酶（SS）在开花后28d内一直维持较高的活性;与此相对应,籽粒蔗糖的含量在开花后28d内呈明显的下降趋势。而旗叶和籽粒中SS活性均与籽粒淀粉的积累速率呈极显著正相关。     

苹果中磷酸蔗糖合酶家族基因的表达特性及其与蔗糖含量的关系

磷酸蔗糖合酶(sucrose phosphate synthase,SPS)是植物中蔗糖合成的主要限速酶,影响植物的生长发育和果实中蔗糖的含量。为探明苹果中SPS基因家族特性及其在蔗糖合成中的作用,该研究从苹果基因组中分离了MdSPS家族基因,分析了它们的进化关系以及mRNA表达特性与酶活性和蔗糖含量的关系。结果显示:(1)在苹果基因组中有8个SPS家族基因表达,它们分别属于双子叶植物的3个SPS亚家族。(2)荧光定量PCR分析显示,苹果C类的MdSPS6基因和A类的MdSPS1a/b基因是苹果中表达丰度最高的SPS基因成员,其中MdSPS6在苹果成熟果中表达丰度最高,其次是成熟叶片,而MdSPS1a/b在不积累蔗糖的幼果中表达丰度最高。(3)在果实发育过程中,除MdSPS1a/b之外,其它5个苹果MdSPS家族基因均随果实的生长表达丰度增加,与SPS活性和蔗糖含量明显呈正相关关系。研究表明,C类家族MdSPS6是苹果果实发育后期和叶片中蔗糖合成的主要SPS基因。     

环糊精葡萄糖基转移酶的基因改造与高效表达

环糊精葡萄糖基转移酶(CGTase,EC 2.4.1.19)是一种多功能酶,主要用于生产环糊精(CD)、糖基化碳水化合物,同时在食品行业也有重要作用。为改善CGTase在这些方面的应用性能,筛选出优势突变酶,异源表达、定点突变、固定化等技术被研究和应用,取得了实质性的进展。综述了CGTase基因高效异源表达策略,概述了基因改造CGTase的研究进展,并且还总结了用于改造CGTase的其他手段,例如固定化酶、嵌合酶、化学添加剂等,以期为在相关CGTase研究领域开展研究提供参考。     

Cyclodextrin glucanotransferase production by cell biocatalysts of alkaliphilic bacilli

Cells of obligated alkaliphiles Bacillus pseudalcaliphilus 20RF and Bacillus pseudalcaliphilus 8SB isolated from Bulgarian habitats, producers of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19), were immobilized by three different techniques: on two types of polysulphone membranes; entrapped in agar-gel beads containing magnetite and by nano-particles of silanized magnetite covalently bound on the cell surface. The biocatalysts obtained demonstrated the opportunity for a significantly enhanced CGTase production compared to free cells for a long period of time (10 days semicontinuous cultivation) without impact on their mechanical stability. The cell membrane-biocatalysts exhibited the highest enzyme activity after 240 h repeated batch cultivation and retained 1.3–2.3-fold increase of the CGTase yield compared to free cells at the end of the process. Membrane biocatalysts were applied for a direct cyclodextrin (CD) production. The results obtained demonstrated the possibility of starch conversion into cyclodextrins by immobilized cells without using of crude or purified enzyme. The membrane biocatalysts of both obligated alkaliphiles formed mainly β- and γ-CDs after 6 h enzyme reaction at pH 9.0 of the reaction mixture. Under these conditions, the quantity of γ-CDs was a relative high, to 35–37% of the total CD amount.     

Effect of organic solvents on enzymatic production of cyclodextrins from unliquefied corn starch in an attrition bioreactor

Cyclodextrins were produced from unliquefied corn starch in the presence of water-miscible organic solvents using cyclodextrin glycosyltransferase (CGTase) in an attrition bioreactor. The production yield was singnificatly increased by isopropanol and tertiary butanol, and maximum enhancement was observed to be about 40% by 5% tertiary butnol. Increase in the production of cyclodextrins by organic solvents seems to be due to the fact that organic solvents decreased the product inhibition of CGTase by forming an inclusion complex with cyclodextrins.     

Magnetic porous corn starch for the affinity purification of cyclodextrin glucanotransferase produced by Bacillus circulans

Magnetic porous corn starch was prepared as an affinity adsorbent for the efficient and simple scale-up procedure for one-step purification of cyclodextrin glucanotransferase (CGTase) from Bacillus circulans. Magnetic affinity separation enabled isolation of CGTase from cultivation media (volumes between 10 and 400 mL) with ca 60–70% recovery after elution with alkaline buffers containing soluble starch; the enzyme purification factor was 19–25 in different batches. The majority of ballast proteins were removed during the purification process, which shows high selectivity of the affinity material used.     

Sucrose phosphorylase of the rumen bacterium Pseudobutyrivibrio ruminis strain A

Aims:  To verify the taxonomic affiliation of bacterium Butyrivibrio fibrisolvens strain A from our collection and to characterize its enzyme(s) responsible for digestion of sucrose.
Methods and Results:  Comparison of the 16S rRNA gene of the bacterium with GenBank showed over 99% sequence identity to the species Pseudobutyrivibrio ruminis . Molecular filtration, native electrophoresis on polyacrylamide gel, zymography and thin layer chromatography were used to identify and characterize the relevant enzyme. An intracellular sucrose phosphorylase with an approximate molecular mass of 52 kDa exhibiting maximum activity at pH 6·0 and temperature 45°C was identified. The enzyme was of inducible character and catalysed the reversible conversion of sucrose to fructose and glucose-1-P. The reaction required inorganic phosphate. The K _m for glucose-1-P formation and fructose release were 3·88 × 10⁻³ and 5·56 × 10⁻³ mol l⁻¹ sucrose, respectively – while the V _max of the reactions were −0·579 and 0·9  μ mol mg protein⁻¹ min⁻¹. The enzyme also released free glucose from glucose phosphate.
Conclusion:  Pseudobutyrivibrio ruminis strain A utilized sucrose by phosphorolytic cleavage.
Significance and Impact of the Study:  Bacterium P. ruminis strain A probably participates in the transfer of energy from dietetary sucrose to the host animal.