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。     

环糊精葡萄糖基转移酶的结构特征与催化机理

随着环糊精在食品、医药等领域的应用越来越广,生产环糊精所必需的环糊精葡萄糖基转移酶(CGT酶)已经成为当今研究的热点。特别是近二十年来,国外对该酶进行了比较深入的研究。首先介绍了CGT酶的功能特性与结构特征。CGT酶是一种多功能型酶,能催化三种转糖基反应(歧化、环化和耦合反应)和水解反应,其中,能将淀粉转化为环糊精的环化反应是特征反应;作为α-淀粉酶家族的成员,CGT酶除了具有与α-淀粉酶相同的A、B、C结构域外,还存在D和E结构域。另外,对CGT酶的催化机理包括底物结合方式、转糖苷反应机理以及环化机理等进行了详细的讨论。     

表面活性剂对大肠杆菌胞外生产α-环糊精葡萄糖基转移酶的影响

研究了不同浓度表面活性剂Tween-80,Triton X-100,SDS对大肠杆菌生产α-环糊精葡萄糖基转移酶（α-CGT酶）的影响。结果表明：发酵初始添加Tween-80和Triton X-100的最适浓度分别为2％,0.5％,最终胞外酶活分别达2.03U/ml和4.92U/ml,相对于未添加表面活性剂时提高4.6倍和12.67倍,且改变添加时间不能提高酶的产量;发酵36 h添加0.02％SDS对α-CGT酶产量促进最大,最终胞外酶活达5.31U/ml,较对照组提高12.75倍。表面活性剂对α-CGT酶生产的促进作用可能是由大肠杆菌细胞内外膜渗透性增加所致,使细胞周质空间中α-CGT酶能更加快速地渗透到胞外。     

化学添加剂提高重组α-环糊精葡萄糖基转移酶酶制剂稳定性

通过向重组α-环糊精葡萄糖基转移酶 (α-CGT酶) 液中添加化学添加剂以提高其热稳定性及贮存稳定性。在不同温度下研究了添加剂对酶液的贮存稳定性影响,并用圆二色谱 (CD) 研究了CGT酶在近紫外区和远紫外区蛋白质结构与热稳定性的变化关系。当单独加入各种添加剂在50 ℃水浴1 h和室温放置108 d后,发现含有20％甘油的酶液稳定性最好,与未加任何添加剂的对照酶液相比仍有91％和50％的酶活,对照酶液在50 ℃水浴1 h后仅有小于10％的活性,室温放置108 d后已经没有酶活。明胶、CaCl2和PEG40     

复合与合成培养基对大肠杆菌胞外生产α-环糊精葡萄糖基转移酶的影响

为实现来源于Paenibacillus macerans JFB05-01的α-环糊精葡萄糖基转移酶（α-CGT酶）的高效胞外表达,以含分泌型信号肽OmpA的大肠杆菌E.coli BL21（DE3）{pET-20b（+）/α-cgt}为研究对象,比较了其在不同诱导条件下复合与合成培养基中生长产酶的规律。结果表明在添加甘氨酸的条件下采用合成培养基,以0.8 g/L/h的乳糖进行流加诱导所得的胞外酶活和生产强度最高。在该条件下发酵30小时后胞外α-CGT酶的环化活性达113.0U/ml（水解活性为79 100.0IU/ml）,是复合培养基胞外产酶的2.3倍,完全满足工业化生产的需求。     

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。     

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．     

利用重组大肠杆菌生产α-环糊精葡萄糖基转移酶

将来源于软化类芽孢杆菌（Paenibacillus macerans）的α-环糊精葡萄糖基转移酶（α-CGT）基因插入含pelB信号肽的质粒pET-20b（＋）中,构建了表达载体pET-20b（＋）／cgt,并将其转化表达宿主E.coli BL21（DE3）。对重组菌E．coli BL21／pET-cgt进行摇瓶发酵条件的优化,确定了其胞外表达α-CGT酶的最适条件：葡萄糖8g/L,乳糖0．5g/L,蛋白胨12g/L,酵母膏24g/L,K2HPO472mmol／L,KH2PO417mmol／L,CaCl2 2．5mmol／L;初始pH为7．0,诱导温度为25℃。在该条件下培养90h后最终α-CGT酶的胞外比活达到22．1u／mL,与来源菌Pmacerans所产天然酶比活相比提高了42倍,实现了α-CGT酶的高效生产。将基因工程菌在上述条件下于3L发酵罐中发酵,90h后胞外酶比活达到22．6U／mL,证实了工业化放大的可能性。     

环糊精葡萄糖基转移酶高效异源表达研究进展

环糊精葡萄糖基转移酶(cyclodextringlycosyltransferase,CGTase)酶法合成环糊精是目前生产环糊精的主要方法。本文介绍了用于生产环糊精葡萄糖基转移酶的几种工程菌株:大肠杆菌、枯草芽孢杆菌以及毕赤酵母,其中大肠杆菌是目前应用最广泛的用于表达CGTase的表达系统。除此之外,本文还总结了高效表达环糊精葡萄糖基转移酶的有效策略:选择合适的表达载体、启动子以及信号肽,以及密码子优化和分子伴侣共表达,以期为在相关CGTase研究领域开展研究提供参考。     

重组β-环糊精葡萄糖基转移酶生产β-环糊精的工艺条件优化

将来自于Bacillus circulans 251的β-CGTase编码基因克隆到表达载体pET-20b(+),转化Escherichia coli BL21(DE3)。经酶活检测培养基上清中的β-CGTase酶活为20 U/mL。对酶转化淀粉生成β-环糊精的反应条件进行了优化,结果表明,当底物马铃薯淀粉浓度15%,反应初始pH5.5,温度30℃,加酶量10 U/g干淀粉,环己烷浓度2.5%-5%(V/V),转化周期24 h,β-环糊精转化率达到最高值75.3%,是国内外报道的酶法生产β-环糊精的最高水平。     

Proteome-based identification of signal peptides for improved secretion of recombinant cyclomaltodextrin glucanotransferase in Escherichia coli

Secretion of recombinant proteins in heterologous host has drawn attention for its simpler purification and downstream processes. Searching for secretion aid molecules to improve protein secretion can be done through synthetic biology, screening of genome data and proteome-based approach. In the present study, the extracellular proteome on starch-containing medium of Bacillus lehensis G1 was analyzed to identify naturally secreted proteins with signal peptide. A total of 87 protein spots were identified by mass spectrometry, which were categorized mostly in the metabolism of carbohydrates and related molecules (20%). Over-expression and secretion studies were performed for all the 14 selected signal peptides fused to a reporter protein, cyclomaltodextrin glucanotransferase (CGTase). All clones were found to allow CGTase to be excreted into the medium, as observed and measured from the iodine plate assay and enzyme activity assay. Compared to native signal peptide (G1) of CGTase, signal peptide of GlcNAc-binding protein A (GAP) significantly improved CGTase activities by 735% and 205% in extracellular and periplasmic compartment, respectively, with an increase of only ∼1.7 fold the amount of β-galactosidase (cell lysis) in the medium. GAP has the highest secretion rate of 45.6 U/ml/h among all clones, where physicochemical characteristics of signal peptide play significant role.     

Substrate complexation and aggregation influence the cyclodextrin glycosyltransferase (CGTase) catalyzed synthesis of alkyl glycosides

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) was used to convert dodecyl-β-maltoside (DDM) to dodecyl-β-maltooctaoside (DDMO) using α-cyclodextrin (α-CD) or starch as glycosyl donors. At 300 mM α-CD, varied DDM concentration and 60 °C, the reaction obeyed Michaelis-Menten kinetics with a K_m value of 18 mM and a V_max value of 100 U/mg enzyme. However, at 25 mM α-CD the reaction rate decreased with increasing DDM concentration (5-50 mM), and when the α-CD concentration was varied at fixed DDM concentration an S shaped curve was obtained. The deviations from Michaelis-Menten kinetics were interpreted as being caused by formation of inclusion complexes between α-CD and DDM and by micellation of DDM. To achieve a high reaction rate, a high concentration of free α-CD is necessary, since α-CD in the form of a complex has low reactivity. When starch is used as glycosyl donor in the CGTase catalyzed alkyl glycoside elongation reaction, it is thus important to choose reaction conditions under which the cyclization of starch to α-CD is efficient.     

Coexpression of folding accessory proteins for production of active cyclodextrin glycosyltransferase of Bacillus macerans in recombinant Escherichia coli

Coexpression of folding accessory proteins, molecular chaperones, and human peptidyl-prolyl cis-trans isomerase (PPIase) increased production of active cyclodextrin glycosyltransferase (CGTase) of Bacillus macerans, which is otherwise mainly expressed as inclusion body in recombinant Escherichia coli. The best partner for soluble expression of CGTase was found to be human PPIase followed by coexpression of DnaK-DnaJ-GrpE together with GroEL-GroES. Such a significant enhancement by human PPIase coexpression seemed to be due to dual functions of chaperone and peptidyl-prolyl cis-trans isomerization. Coexpression of GroEL-GroES or minichaperone alone did not influence the specific CGTase activity. For production of active CGTase in large amounts, a high cell density culture was achieved using a pH-stat fed-batch strategy. The optimized fed-batch fermentation resulted in dry cell weight of 103.4 g/L and CGTase activity of 1200 U/mL. Combination of human PPIase expression at a gene level and cell culture optimization at a process scale exerted a synergistic effect on the product yield of soluble CGTase expression in recombinant E. coli.     

Optimization of defined medium for recombinant Komagataella phaffii expressing cyclodextrin glycosyltransferase

The cyclodextrin glycosyltransferase (CGTase) is an important enzyme for cyclodextrin (CD) production, and is also widely used in the biotechnology, food, and pharmaceuticals industries. Secretory CGTase production by recombinant Komagataella phaffii using defined medium is a promising approach because of low cost, less impurity protein. It was found that no CGTase was expressed using traditional defined medium (basal salt medium [BSM]) because of pH value decreasing significantly. CGTase was expressed by recombinant K. phaffii through pH maintenance in range of 5.5–7.0. β-CGTase activity increased to 122.0 U/mL after optimization of glycerol, phosphate buffer, pH value, ammonium sulfate, temperature, methanol, and additives based on BSM, establishing a modified defined medium. These results showed that it was necessary to establish recombinant K. phaffii-based special defined medium although the same host cell used for different heterologous protein expression.     

Recent advances in discovery,heterologous expression,and molecular engineering of cyclodextrin glycosyltransferase for versatile applications

Cyclodextrin glycosyltransferase (CGTase) is an important enzyme with multiple functions, in particular the production of cyclodextrins. It is also widely applied in baking and carbohydrate glycosylation because it participates in various types of catalytic reactions. New applications are being found with novel CGTases being isolated from various organisms. Heterologous expression is performed for the overproduction of CGTases to meet the requirements of these applications. In addition, various directed evolution techniques have been applied to modify the molecular structure of CGTase for improved performance in industrial applications. In recent years, substantial progress has been made in the heterologous expression and molecular engineering of CGTases. In this review, we systematically summarize the heterologous expression strategies used for enhancing the production of CGTases. We also outline and discuss the molecular engineering approaches used to improve the production, secretion, and properties (e.g., product and substrate specificity, catalytic efficiency, and thermal stability) of CGTase.     

Comparison of different signal peptides for protein secretion in nonlytic insect cell system

Protein expression and secretion in insect cells have been widely studied in the baculovirus-infected insect cell system. In directly transfected insect cells only intracellular expression and purification of recombinant proteins have been studied in detail. To examine multiple recombinant protein variants, easy and fast expression and a purification screening system are required. The aim of this study was to establish an effective and rapid secretion system for human azurocidin using directly transfected insect cells. We also constructed and tested expression vectors possessing heterologous signal peptides derived from human azurocidin, yellow lupin diphosphonucleotide phosphatase/phosphodiesterase (PPD1), and papaya papain IV to secrete yellow lupin and red kidney bean purple acid phosphatases, PPD1, and papain IV. Our results demonstrate that the secretion vectors used here can direct recombinant proteins to the culture medium very effectively, allowing their simple purification on a small/medium scale. Based on secretion and activity analyses it seems that the azurocidin signal peptide is one of the most potent secretion signals.     

Secretion of xylanase A2 in Streptomyces lividans: dependence on signal peptides length, number and composition

The signal peptide (sp) in Streptomyces lividans xylanase A2 (XlnA2) was replaced by sps containing, in frame in their sequences, one, two, three or four initiation codons, each preceded by a Shine-Dalgarno (SD) sequence. Precursors of the corresponding proteins should thus have sps of, respectively, 27, 46, 82 and 91 amino acids (aa) long. By radiolabelling of S. lividans harboring the different constructs inserted in a multicopy plasmid and by immunoprecipitation with anti-xylanase antibodies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation, precursors of the expected sizes were obtained in each clone. This indicates that ribosomes can synthesize different XlnA2 precursors from initiation codons inserted in the sp sequence, independently of their number. The amount of these synthesized precursors was also shown to be inversely proportional to their length when comparing the specific activity of labelling versus sp length. In clones producing more than one precursor, a smear appeared on the autoradiograms, suggesting some degree of precursor degradation. As determined by pulse-chase experiments, the rate of disappearance was almost the same for precursors of different lengths, but this might be the result of both true processing and proteolytic degradation. Furthermore, S. lividans rapidly degraded XlnA2 either when deprived of its sp or in the absence of the signal peptidase cleavage site.