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。     

Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications

Cyclodextrin glucanotransferases (CGTases) are industrially important enzymes that produce cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. Cyclodextrin glucanotransferases are also applied as catalysts in the synthesis of glycosylated molecules and can act as antistaling agents in the baking industry. To improve the performance of CGTases in these various applications, protein engineers are screening for CGTase variants with higher product yields, improved CD size specificity, etc. In this review, we focus on the strategies employed in obtaining CGTases with new or enhanced enzymatic capabilities by searching for new enzymes and improving existing enzymatic activities via protein engineering.     

Relation between domain evolution, specificity, and taxonomy of the alpha-amylase family members containing a C-terminal starch-binding domain.

The alpha-amylase family (glycoside hydrolase family 13; GH 13) contains enzymes with approximately 30 specificities. Six types of enzyme from the family can possess a C-terminal starch-binding domain (SBD): alpha-amylase, maltotetraohydrolase, maltopentaohydrolase, maltogenic alpha-amylase, acarviose transferase, and cyclodextrin glucanotransferase (CGTase). Such enzymes are multidomain proteins and those that contain an SBD consist of four or five domains, the former enzymes being mainly hydrolases and the latter mainly transglycosidases. The individual domains are labelled A [the catalytic (beta/alpha)8-barrel], B, C, D and E (SBD), but D is lacking from the four-domain enzymes. Evolutionary trees were constructed for domains A, B, C and E and compared with the 'complete-sequence tree'. The trees for domains A and B and the complete-sequence tree were very similar and contain two main groups of enzymes, an amylase group and a CGTase group. The tree for domain C changed substantially, the separation between the amylase and CGTase groups being shortened, and a new border line being suggested to include the Klebsiella and Nostoc CGTases (both four-domain proteins) with the four-domain amylases. In the 'SBD tree' the border between hydrolases (mainly alpha-amylases) and transglycosidases (principally CGTases) was not readily defined, because maltogenic alpha-amylase, acarviose transferase, and the archaeal CGTase clustered together at a distance from the main CGTase cluster. Moreover the four-domain CGTases were rooted in the amylase group, reflecting sequence relationships for the SBD. It appears that with respect to the SBD, evolution in GH 13 shows a transition in the segment of the proteins C-terminal to the catalytic (beta/alpha)8-barrel(domain A).     

Analysis of mutations in cyclodextrin glucanotransferase from Bacillus stearothermophilus which affect cyclization characteristics and thermostability.

Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) produces cyclodextrin from starch. The CGTase molecule is composed of four globular domains, A, B, C, and D. In order to gain better understanding of the amylolytic and cyclization mechanisms of CGTase, mutant CGTases were constructed from a CGTase gene (cgt1) of Bacillus stearothermophilus NO2. Cgt1-F191Y (Phe at position 191 was replaced by Tyr), Cgt1-F191Y-F255Y, Cgt1-W254V-F255I, Cgt1-W254V, and Cgt1-F255I were constructed for the analysis of the NH2-terminal region. It was revealed that amino acids surrounding a spiral amylose are important for cyclization characteristics and that hydrophobic amino acids just after the Glu catalytic site play an important role in the hydrolysis characteristics of the enzyme. Mutant CGTases Cgt1-T591F and Cgt1-W629F were also constructed to study the role of a second substrate-binding site in domain D, and it was suggested that substrate binding at both domains A and D stabilized the enzyme and optimized cyclodextrin production.     

Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase alpha-cyclodextrin production

Cyclodextrin glycosyltransferases (CGTase) (EC 2.4.1.19) are extracellular bacterial enzymes that generate cyclodextrins from starch. All known CGTases produce mixtures of alpha, beta, and gamma-cyclodextrins. A maltononaose inhibitor bound to the active site of the CGTase from Bacillus circulans strain 251 revealed sugar binding subsites, distant from the catalytic residues, which have been proposed to be involved in the cyclodextrin size specificity of these enzymes. To probe the importance of these distant substrate binding subsites for the alpha, beta, and gamma-cyclodextrin product ratios of the various CGTases, we have constructed three single and one double mutant, Y89G, Y89D, S146P and Y89D/S146P, using site-directed mutagenesis. The mutations affected the cyclization, coupling; disproportionation and hydrolyzing reactions of the enzyme. The double mutant Y89D/S146P showed a twofold increase in the production of alpha-cyclodextrin from starch. This mutant protein was crystallized and its X-ray structure, in a complex with a maltohexaose inhibitor, was determined at 2.4 A resolution. The bound maltohexaose molecule displayed a binding different from the maltononaose inhibitor, allowing rationalization of the observed change in product specificity. Hydrogen bonds (S146) and hydrophobic contacts (Y89) appear to contribute strongly to the size of cyclodextrin products formed and thus to CGTase product specificity. Changes in sugar binding subsites -3 and -7 thus result in mutant proteins with changed cyclodextrin production specificity.     

Hydrophobic interaction chromatography on octyl sepharose--an approach for one-step platform purification of cyclodextrin glucanotransferases

Cyclodextrin glucanotransferase (CGTase) from Bacillus circulans ATCC 21783 was concentrated by ultrafiltration and subsequently purified by hydrophobic interaction chromatography on Octyl Sepharose 4 fast flow. The matrix was able to bind selectively to the enzyme at a very low ammonium sulfate concentration of 0.67 M and enzyme desorption was performed by decreasing gradient of the salt. The overall recovery was 80% with 689-fold purity. CGTases derived from four soil isolates and Toruzyme, the commercial preparation of CGTase, also bound to Octyl Sepharose under similar conditions at 0.67 M and eluted at 0.55-0.5 M of ammonium sulfate. Octyl Sepharose chromatography can thus be used as a platform approach for purification of CGTases from various bacterial sources. Long stretches of sequence predominated by hydrophobic amino acids are reportedly present in the starch binding domains of CGTases. Starch binding experiments indicated the binding of the enzymes to the octyl matrix through these domains.     

CGTase,a novel antimicrobial protein from Bacillus cereus YUPP-10, suppresses Verticillium dahliae and mediates plant defence responses

Verticillium wilt is a plant vascular disease caused by the soilborne fungus Verticillium dahliae that severely limits cotton production. In a previous study, we screened Bacillus cereus YUPP-10, an efficient antagonistic bacterium, to uncover mechanisms for controlling verticillium wilt. Here, we report a novel antimicrobial cyclodextrin glycosyltransferase (CGTase) from YUPP-10. Compared to other CGTases, six different conserved domains were identified, and six mutants were constructed by gene splicing with overlap extension PCR. Functional analysis showed that domain D was important for hydrolysis activity and domains A1 and C were important for inducing disease resistance. Direct effects of recombinant CGTase on V. dahliae included reduced mycelial growth, spore germination, spore production, and microsclerotia germination. In addition, CGTase also elicited cotton's innate defence reactions. Transgenic Arabidopsis thaliana lines that overexpress CGTase showed higher resistance to verticillium wilt. Transgenic CGTase A. thaliana plants grew faster and resisted disease better. CGTase overexpression enabled a burst of reactive oxygen species production and activated pathogenesis-related gene expression, indicating that the transgenic cotton was better prepared to protect itself from infection. Our work revealed that CGTase could inhibit the growth of V. dahliae, activate innate immunity, and play a major role in the biocontrol of fungal pathogens.     

HYDROPHOBIC INTERACTION CHROMATOGRAPHY ON OCTYL SEPHAROSE—AN APPROACH FOR ONE-STEP PLATFORM PURIFICATION OF CYCLODEXTRIN GLUCANOTRANSFERASES

Cyclodextrin glucanotransferase (CGTase) from Bacillus circulans ATCC 21783 was concentrated by ultrafiltration and subsequently purified by hydrophobic interaction chromatography on Octyl Sepharose 4 fast flow. The matrix was able to bind selectively to the enzyme at a very low ammonium sulfate concentration of 0.67 M and enzyme desorption was performed by decreasing gradient of the salt. The overall recovery was 80% with 689-fold purity. CGTases derived from four soil isolates and Toruzyme, the commercial preparation of CGTase, also bound to Octyl Sepharose under similar conditions at 0.67 M and eluted at 0.55–0.5 M of ammonium sulfate. Octyl Sepharose chromatography can thus be used as a platform approach for purification of CGTases from various bacterial sources. Long stretches of sequence predominated by hydrophobic amino acids are reportedly present in the starch binding domains of CGTases. Starch binding experiments indicated the binding of the enzymes to the octyl matrix through these domains.     

Improved thermostability of bacillus circulans cyclodextrin glycosyltransferase by the introduction of a salt bridge

Cyclodextrin glycosyltransferase (CGTase) catalyzes the formation of cyclodextrins from starch. Among the CGTases with known three-dimensional structure, Thermoanaerobacterium thermosulfurigenes CGTase has the highest thermostability. By replacing amino acid residues in the B-domain of Bacillus circulans CGTase with those from T. thermosulfurigenes CGTase, we identified a B. circulans CGTase mutant (with N188D and K192R mutations), with a strongly increased activity half-life at 60 degrees C. Asp188 and Arg192 form a salt bridge in T. thermosulfurigenes CGTase. Structural analysis of the B. circulans CGTase mutant revealed that this salt bridge is also formed in the mutant. Thus, the activity half-life of this enzyme can be enhanced by rational protein engineering.     

Transglycosylation of L-ascorbic acid

Cyclodextrin glucanotransferases (CGTase, EC 2.4.1.19) produced by mesophilic, thermophilic, and halophilic bacilli, as well as maltase (EC 3.2.1.20) produced by various strains of Saccharomyces cerevisiae have been applied for transglycosylation of L-ascorbic acid using starch, maltodextrin, gamma-cyclodextrin, and maltose as donors of glucosyl residue. The CGTases produced by thermophilic strains are the most efficient. The degree of transglucosylation is more than 60%.     

Iterative Saturation Mutagenesis of ?6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2-O-d-Glucopyranosyl-l-Ascorbic Acid Synthesis

2-O-d-Glucopyranosyl-l-ascorbic acid (AA-2G), a stable l-ascorbic acid derivative, is usually synthesized by cyclodextrin glycosyltransferase (CGTase), which contains nine substrate-binding subsites (from +2 to −7). In this study, iterative saturation mutagenesis (ISM) was performed on the −6 subsite residues (Y167, G179, G180, and N193) in the CGTase from Paenibacillus macerans to improve its specificity for maltodextrin, which is a cheap and easily soluble glycosyl donor for AA-2G synthesis. Site saturation mutagenesis of four sites—Y167, G179, G180, and N193—was first performed and revealed that four mutants—Y167S, G179R, N193R, and G180R—produced AA-2G yields higher than those of other mutant and wild-type CGTases. ISM was then conducted with the best positive mutant as a template. Under optimal conditions, mutant Y167S/G179K/N193R/G180R produced the highest AA-2G titer of 2.12 g/liter, which was 84% higher than that (1.15 g/liter) produced by the wild-type CGTase. Kinetics analysis of AA-2G synthesis using mutant CGTases confirmed the enhanced maltodextrin specificity and showed that compared to the wild-type CGTase, the mutants had no cyclization activity but high hydrolysis and disproportionation activities. A possible mechanism for the enhanced substrate specificity was also analyzed through structure modeling of the mutant and wild-type CGTases. These results indicated that the −6 subsite played crucial roles in the substrate binding and catalytic reactions of CGTase and that the obtained CGTase mutants, especially Y167S/G179K/N193R/G180R, are promising starting points for further development through protein engineering.     

Optimisation of batch culture conditions for cyclodextrin glucanotransferase production from Bacillus circulans DF 9R

Background  

The extracellular enzyme cyclodextrin glucanotransferase (CGTase) synthesizes cyclic malto-oligosaccharides called cyclodextrins (CDs) from starch and related α-1,4-glucans. CGTases are produced by a variety of bacteria, mainly Bacillus species, by submerged culture in complex medium. CGTases differ in the amount and types of CDs produced. In addition, CGTase production is highly dependent on the strain, medium composition and culture conditions. Therefore we undertook this study with a newly isolated strain of Bacillus circulans.     

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。     

New Applications for Phage Integrases

Within the last 25 years, bacteriophage integrases have rapidly risen to prominence as genetic tools for a wide range of applications from basic cloning to genome engineering. Serine integrases such as that from ?C31 and its relatives have found an especially wide range of applications within diverse micro-organisms right through to multi-cellular eukaryotes. Here, we review the mechanisms of the two major families of integrases, the tyrosine and serine integrases, and the advantages and disadvantages of each type as they are applied in genome engineering and synthetic biology. In particular, we focus on the new areas of metabolic pathway construction and optimization, biocomputing, heterologous expression and multiplexed assembly techniques. Integrases are versatile and efficient tools that can be used in conjunction with the various extant molecular biology tools to streamline the synthetic biology production line.     

Microbial xylanases: Engineering, production and industrial applications

Enzymatic depolymerization of hemicellulose to monomer sugars needs the synergistic action of multiple enzymes, among them endo-xylanases (EC 3.2.1.8) and β-xylosidases (EC 3.2.1.37) (collectively xylanases) play a vital role in depolymerizing xylan, the major component of hemicellulose. Recent developments in recombinant protein engineering have paved the way for engineering and expressing xylanases in both heterologous and homologous hosts. Functional expression of endo-xylanases has been successful in many hosts including bacteria, yeasts, fungi and plants with yeasts being the most promising expression systems. Functional expression of β-xylosidases is more challenging possibly due to their more complicated structures. The structures of endo-xylanases of glycoside hydrolase families 10 and 11 have been well elucidated. Family F/10 endo-xylanases are composed of a cellulose-binding domain and a catalytic domain connected by a linker peptide with a (β/α)(8) fold TIM barrel. Family G/11 endo-xylanases have a β-jelly roll structure and are thought to be able to pass through the pores of hemicellulose network owing to their smaller molecular sizes. The structure of a β-d-xylosidase belonging to family 39 glycoside hydrolase has been elucidated as a tetramer with each monomer being composed of three distinct regions: a catalytic domain of the canonical (β/α)(8) - TIM barrel fold, a β-sandwich domain and a small α-helical domain with the enzyme active site that binds to d-xylooligomers being present on the upper side of the barrel. Glycosylation is generally considered as one of the most important post-translational modifications of xylanases, but a few examples showed functional expression of eukaryotic xylanases in bacteria. The optimal ratio of these synergistic enzymes is very important in improving hydrolysis efficiency and reducing enzyme dosage but has hardly been addressed in literature. Xylanases have been used in traditional fields such as food, feed and paper industries for a longer time but more and more attention has been paid to using them in producing sugars and other chemicals from lignocelluloses in recent years. Mining new genes from nature, rational engineering of known genes and directed evolution of these genes are required to get tailor-made xylanases for various industrial applications.     

Altered product specificity of a cyclodextrin glycosyltransferase by molecular imprinting with cyclomaltododecaose

Cyclodextrin glycosyltransferases (CGTases), members of glycoside hydrolase family 13, catalyze the conversion of amylose to cyclodextrins (CDs), circular α‐(1,4)‐linked glucopyranose oligosaccharides of different ring sizes. The CD containing 12 α‐D‐glucopyranose residues was preferentially synthesized by molecular imprinting of CGTase from Paenibacillus sp. A11 with cyclomaltododecaose (CD₁₂) as the template molecule. The imprinted CGTase was stabilized by cross‐linking of the derivatized protein. A high proportion of CD₁₂ and larger CDs was obtained with the imprinted enzyme in an aqueous medium. The molecular imprinted CGTase showed an increased catalytic efficiency of the CD₁₂‐forming cyclization reaction, while decreased k_cat/K_m values of the reverse ring‐opening reaction were observed. The maximum yield of CD₁₂ was obtained when the imprinted CGTase was reacted with amylose at 40°C for 30 min. Molecular imprinting proved to be an effective means toward increase in the yield of large‐ring CDs of a specific size in the biocatalytic production of these interesting novel host compounds for molecular encapsulations. Copyright © 2010 John Wiley & Sons, Ltd.     

Enzymatic production of cyclodextrins

Cyclodextrins (CD) are enzymatically modified starches with a wide range of applications in food, pharmaceutical and chemical industries, agriculture and environmental engineering. They are produced from starch via enzymatic conversion using cyclodextrin glycosyl transferases (CGTases) and partly alpha-amylases. Due to its low solubility in water, separation and purification of beta-CD is relatively easy compared to alpha- and gamma-CD. In recent years more economic processes for gamma-CD and especially alpha-CD production have been developed using improved CGTases and downstream processing. New purification steps, e.g. affinity adsorption, may reduce the use of complexing agents. The implementation of thermostable CGTases can simplify the production process and increase the selectivity of the reaction. A tabular overview of alpha-CD production processes is presented.     

Sequencing,cloning, and heterologous expression of cyclomaltodextrin glucanotransferase of Bacillus firmus strain 37 in Bacillus subtilis WB800

Bacillusfirmus strain 37 produces the cyclomaltodextrin glucanotransferase (CGTase) enzyme and CGTase produces cyclodextrins (CDs) through a starch cyclization reaction. The strategy for the cloning and expression of recombinant CGTase is a potentially viable alternative for the economically viable production of CGTase for use in industrial processes. The present study used Bacillus subtilis WB800 as a bacterial expression host for the production of recombinant CGTase cloned from the CGTase gene of B. firmus strain 37. The CGTase gene was cloned in TOPO-TA^® plasmid, which was transformed in Escherichia coli DH5α. The subcloning was carried out with pWB980 plasmid and transformation in B. subtilis WB800. The 2xYT medium was the most suitable for the production of recombinant CGTase. The enzymatic activity of the crude extract of the recombinant CGTase of B. subtilis WB800 was 1.33 µmol β-CD/min/mL, or 7.4 times greater than the enzymatic activity of the crude extract of CGTase obtained from the wild strain. Following purification, the recombinant CGTase exhibited an enzymatic activity of 157.78 µmol β-CD/min/mL, while the activity of the CGTase from the wild strain was 9.54 µmol β-CD/min/mL. When optimal CDs production conditions for the CGTase from B. firmus strain 37 were used, it was observed that the catalytic properties of the CGTase enzymes were equivalent. The strategy for the cloning and expression of CGTase in B. subtilis WB800 was efficient, with the production of greater quantities of CGTase than with the wild strain, offering essential data for the large-scale production of the recombinant enzyme.

     

Effects of essential carbohydrate/aromatic stacking interaction with Tyr100 and Phe259 on substrate binding of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp. 1011

The stacking interaction between a tyrosine residue and the sugar ring at the catalytic subsite -1 is strictly conserved in the glycoside hydrolase family 13 enzymes. Replacing Tyr100 with leucine in cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. 1011 to prevent stacking significantly decreased all CGTase activities. The adjacent stacking interaction with both Phe183 and Phe259 onto the sugar ring at subsite +2 is essentially conserved among CGTases. F183L/F259L mutant CGTase affects donor substrate binding and/or acceptor binding during transglycosylation [Nakamura et al. (1994) Biochemistry 33, 9929-9936]. To elucidate the precise role of carbohydrate/aromatic stacking interaction at subsites -1 and +2 on the substrate binding of CGTases, we analyzed the X-ray structures of wild-type (2.0 A resolution), and Y100L (2.2 A resolution) and F183L/F259L mutant (1.9 A resolution) CGTases complexed with the inhibitor, acarbose. The refined structures revealed that acarbose molecules bound to the Y100L mutant moved from the active center toward the side chain of Tyr195, and the hydrogen bonding and hydrophobic interaction between acarbose and subsites significantly diminished. The position of pseudo-tetrasaccharide binding in the F183L/F259L mutant was closer to the non-reducing end, and the torsion angles of glycosidic linkages at subsites -1 to +1 on molecule 1 and subsites -2 to -1 on molecule 2 significantly changed compared with that of each molecule of wild-type-acarbose complex to adopt the structural change of subsite +2. These structural and biochemical data suggest that substrate binding in the active site of CGTase is critically affected by the carbohydrate/aromatic stacking interaction with Tyr100 at the catalytic subsite -1 and that this effect is likely a result of cooperation between Tyr100 and Phe259 through stacking interaction with substrate at subsite +2.