Characterisation of a thermoalkali-stable cyclodextrin glycosyltransferase from the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii

The thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii produces an extracellular CGTase when grown on starch at 55°C and pH 9.0. The gene encoding this CGTase was cloned and successfully expressed in Escherichia coli. It encodes a protein consisting of 721 amino acids with a signal sequence of 34 amino acids. On SDS–polyacrylamide gels, the purified CGTase from A. gottschalkii displayed the expected molecular mass of 78 kDa. The recombinant enzyme was purified with a yield of 13.5% and displayed a specific activity of 210 units/mg. This CGTase, which represents the first report of a CGTase from an anaerobic thermoalkaliphile, was active at a broad range of temperature and pH, namely 55–70°C and pH 5–10. It completely converted amylose, amylopectin and native starch to cyclodextrins, preferentially -cyclodextrin. With a longer incubation period, the -cyclodextrin to -cyclodextrin ratio declined. Variations in substrate type and concentration influenced the product pattern. Increasing the substrate concentration (0.5–20.0%) and glucans containing branching points (-1,6 glycosidic linkages) shifted the product pattern to: -cyclodextin > -cyclodextrin > -cyclodextrin. In addition to these cyclodextrins, larger cyclodextrins (>8 glucose units) were formed in the initial reaction period. The CGTase was stabilised against thermal inactivation by calcium ions and high substrate concentrations; and 5 mM of CaCl₂ shifted the apparent melting point of the enzyme from 60°C to 69°C.Dedicated to Prof. Dr. Hans G. Schlegel on the occasion of his 80th birthday.     

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。     

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.     

Mass transfer studies on the reduction of Cr(VI) using calcium alginate immobilized Bacillus sp. in packed bed reactor

This study presents the external mass transfer effects on the reduction of hexavalent chromium (Cr(VI)) using calcium alginate immobilized Bacillus sp. in a re-circulated packed bed batch reactor (RPBR). The effect of flow rate on the reduction Cr(VI) was studied. Theoretically calculated rate constants for various flow rates were analyzed using external film diffusion models and compared with experimental values. The external mass transfer coefficients for the bioconversion of Cr(VI) were also investigated. The external mass transfer effect was correlated with a model of the type J_D = K Re⁻⁽¹⁻ⁿ⁾. The model was tested with various K values and the mass transfer correlation J_D = 5.7 Re^−0.70 was found to predict the experimental data accurately. The proposed model would be useful for the design of industrial reactor and scale up.     

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.     

Production of Schardinger beta-dextrin by soluble and immobilized cyclodextrin glycosyltransferase of an alkalophilic Bacillus sp.

Succinylated cyclodextrin glycosyltransferase (EC 3.2.1.19) of an alkalophilic Bacillus sp. was adsorbed on a vinylpyridine copolymer. The enzyme had about 25% of the activity of soluble enzyme added. No increase of pH or thermal stability of the enzyme was observed by the adsorption, whereas optimum temperature for the enzyme action was shifted from 50 to 55 degrees C. The enzyme converted starch to cyclodextrine without significant loss of activity under the conditions of 4 times reusing of 6 hr conversion by the batch system or 2 weeks continuous reaction by the column system at 55 degrees C and pH 8.0. About 46% of the potato starch solution [15% (w/v)] was converted to cyclodextrins by the enzyme, and 52% was converted by the simultaneous action of the enzyme and alkaline pullulanase of alkalophilic Bacillus sp. (No. 202-1). These values were almost the same as those obtained by the soluble enzyme or enzymes system.     

信号肽及化学通透剂对环糊精葡萄糖基转移酶胞外分泌的影响

【目的】研究不同的信号肽和化学通透剂对重组环糊精葡萄糖基转移酶(CGTase)胞外分泌的影响,提高CGTase的胞外分泌量。【方法】扩增地芽孢杆菌CHB1(Geobacillus sp.CHB1)的CGTase基因,构建带有地芽孢杆菌CHB1自身信号肽、Omp A、Pel B信号肽和不带信号肽的4种重组质粒;比较4种重组质粒对重组CGTase胞外分泌的影响,筛选最优的信号肽;考察甘氨酸、Triton X-100、SDS和Tween 80四种化学通透剂对重组CGTase胞外分泌的影响,确定最佳的化学通透剂及其浓度。【结果】Omp A信号肽介导的分泌效果最好,胞外酶活达到7.44 U/m L,分别是Pel B、CHB1信号肽的2.04倍和11.27倍,不带信号肽的重组质粒菌胞外检测不到酶活;携带Omp A信号肽的重组质粒菌发酵48 h,同时添加浓度为0.6%的甘氨酸和0.3%的Triton X-100,胞外酶活达最大到14.27 U/m L;SDS和Tween 80对该酶的胞外分泌具有明显的抑制作用。【结论】Omp A信号肽的介导效果最佳,同时添加浓度为0.6%和0.3%的甘氨酸和Triton X-100可以有效促进胞外分泌,为该重组酶的高效胞外分泌提供了一种有效的方法。     

Stability and activity of cross-linking enzyme crystals of cyclodextrin glucanotransferase isolated from Bacillus macerans

Recently, a new technique using cross-linking enzyme crystals (CLECs) was introduced in the field of enzyme technology. CLECs are solid crystalline particles which are insoluble in both water and organic solvents. Chemical cross-linking of the enzyme crystals preserves the catalytic activity even in harsh conditions such as at high temperature, at extreme pH, in organic solvents, and in the presence of proteases and radical chemicals. CLECs of cyclodextrin glucanotransferase (CGTase) could be useful biocatalysts because they were stable at elevated temperature, in organic solvents, and in the presence of enzyme inactivation surfactant. They also maintained their activity against protein-digesting enzyme.     

Production of cyclodextrin from starch by cyclodextrin glycosyltransferase from Bacillus firmus and characterization of purified enzyme

During screening for cyclodextrin-forming microorganisms, an alkalophilic Bacillus sp, which produced high activity of cyclodextrin glycosyltransferase, was isolated and identified as Bacillus firmus. The crude enzyme transformed starch to mainly β-and γ-cyclodextrin. The purified enzyme had an optimum pH of 7.5–8.5 and its optimum temperature was 65°C, which is the highest optimum temperature as compared to other cyclodextrin glycosyltransferases except that produced by Bacillus amyloliquefaciens. Received 06 January 1997/ Accepted in revised form 20 March 1997     

纳米材料固定化酶的研究进展

近年来,纳米技术为酶固定化提供了多种纳米级材料,纳米材料固定化酶不仅具有高的酶负载量,而且具有良好的酶稳定性。本文基于纳米材料固定化酶,对纳米材料的种类进行了总结,分析了纳米材料对固定化酶性能的影响,并介绍了纳米级固定化方法及纳米材料固定化酶在生物转化、生物传感器、生物燃料电池等领域的应用。     

Continuous production of fructose-syrups from inulin by immobilized inulinase from recombinantSaccharomyces cerevisiae

Recombinant exoinulinase was partially purified from the culture supernatant ofS. cerevisiae by (NH₄)₂SO₄ precipitation and PEG treatment. The purified inulinase was immobilized onto Amino-cellulofine with glutaraldehyde as a cross-linking agent. Immobilization yield based on the enzyme activity was about 15%. Optimal pH and temperature of immobilized enzyme were found to be 5.0 and 60°C, respectively. The enzyme activity was stably maintained in the pH ranges of 4.5 to 6.0 at 60°C. 100% of enzyme activity was observed even after incubation for 24 hr at 60°C. In the operation of a packed-bed reactor containing 412 U inulinase, dahalia inulin of 7.5%(w/v) concentration was completely hydrolyzed at flow rate of 2.0 mL/min at 60°C, resulting in a volumetric productivity of 693 g-reducing sugars/L/h. Under the reaction conditions of 1.0 mL/min flow rate with 2.5% inulin at 60°C, the reactor was successfully operated over 30 days without loss of inulinase activity.     

Biocatalytic asymmetric hydroxylation of hydrocarbons by free and immobilized Bacillus megaterium cells

Screening of soil bacteria with allylbenzene resulted in a Bacillus megaterium strain, which hydroxylates simple hydrocarbons in high enantiomeric excess (ee up to 99%). Benzylic and nonbenzylic hydroxylation products were obtained, without the usually observed high preference for the benzylic position. The immobilization of the B. megaterium cells in alginate gel effectively improved the stability of the cells and increased the amounts of products formed, without loss of enantioselectivity. The product ratio ( vs. β hydroxylation) was shifted towards benzylic hydroxylation, which suggests that at least two hydroxylating enzymes with distinct regioselectivity are involved. Comparison to free-cell fermentations in small- and large-scale bioreactors (up to 2000 ml) showed that the use of immobilized cells is advantageous, as they are easier to handle and yield higher amounts of oxidation products.     

Immobilization of Acidithiobacillus ferrooxidans by a PVA–boric acid method for ferrous sulphate oxidation

Acidithiobacillus ferrooxidans was immobilized in poly(vinyl alcohol) (PVA) by a PVA–boric acid method, and spherical beads of uniform size were produced. Biooxidation of ferrous iron by immobilized cells was investigated in repeated batch culture and continuous operation in a laboratory scale packed-bed bioreactor. During repeated batch culture, the cell-immobilized gels were stable and showed high constant iron-oxidizing activity. In continuous operation in a packed-bed bioreactor, biooxidation of ferrous iron fits a plug-flow reaction model well. A maximum Fe²⁺ oxidation rate of 1.89 g l⁻¹ h⁻¹ was achieved at the dilution rate of 0.38 h⁻¹ or higher, while no obvious precipitate was detected in the bioreactor.     

Production of cyclodextrin glycosyltransferase by immobilized <Emphasis Type="Italic">Bacillus</Emphasis> sp. on chitosan matrix

The whole-cell immobilization on chitosan matrix was evaluated. Bacillus sp., as producer of CGTase, was grown in solid-state and batch cultivation using three types of starches (cassava, potato and cornstarch). Biomass growth and substrate consumption were assessed by flow cytometry and modified phenol–sulfuric acid assays, respectively. Qualitative analysis of CGTase production was determined by colorless area formation on solid culture containing phenolphthalein. Scanning electron microscopy (SEM) analysis demonstrated that bacterial cells were immobilized on chitosan matrix efficiently. Free cells reached very high numbers during batch culture while immobilized cells maintained initial inoculum concentration. The maximum enzyme activity achieved by free cells was 58.15 U ml^?1 (36 h), 47.50 U ml^?1 (36 h) and 68.36 U ml^?1 (36 h) on cassava, potato and cornstarch, respectively. CGTase activities for immobilized cells were 82.15 U ml^?1 (18 h) on cassava, 79.17 U ml^?1 (12 h) on potato and 55.37 U ml^?1 (in 6 h and max 77.75 U ml^?1 in 36 h) on cornstarch. Application of immobilization technique increased CGTase activity significantly. The immobilized cells produced CGTase with higher activity in a shorter fermentation time comparing to free cells.     

Resolution of 2-octanol by SBA-15 immobilized Pseudomonas sp. lipase

Lipase from Pseudomonas sp. (PSL) was immobilized on SBA-15 (a highly ordered hexagonal array mesoporous silica molecular sieve) through physical adsorption and the immobilized PSL was used in resolution of (R,S)-2-octanol with vinyl acetate as acyl donor. Enhanced activity and enantioselectivity were observed for the immobilized PSL compared with those of the free one. The effects of reaction conditions, such as solvents, temperature, water activity and substrate ratio were investigated. Under the optimum conditions, the residual (S)-2-octanol was recovered with 99% enantiomeric excess at 52% conversion. The results also indicated that the immobilized PSL maintained 90% of its initial activity even after reusing it five times.     

Improvement of cyclodextrin glycosyltransferase (CGTase) production by recombinant Escherichia coli pAD26 immobilized on the cotton

The cyclodextrin glycosyltransferase (CGTase) of the recombinants Escherichia coli pAD26 cells immobilized on cotton was optimally produced by statistical methodology. Primarily, carbon and nitrogen sources were selected by one-factor-at-a-time method. Wheat starch, Casamino acid, Edamin and Hy-soy were identified as the best nutrients. These sources were secondly confirmed by Plackett-Burman design (fifteen variables were studied with sixteen experiments), as the most significant components with respect to CGTase production. In the third step, concentration of most significant factors and their interaction were optimized with a Box-Behnken experimental design. Under the optimized conditions (agitation 200 rpm, yeast extract concentration 20 g/L, wheat starch concentration 10 g/L and Hy-soy concentration 2.5 g/L), CGTase yield 145.11 U/mL was 3.6 and 23 folds higher than those obtained by the use of the initial conditions (39.77 U/mL) and free cells (6.37 U/mL), respectively.     

Immobilized Cyclodextrin Glycosyl Transferase for the Continuous Production of Cyclodextrins

Cyclodextrin glycosyl transferase (E.C: 2.4.1.19) from Bacillus, macerans and from a Bacillus sp. isolate was immobilized by two methods, viz. to epoxy-activated Sepharose and to alkylamine silica treated with glutaraldehyde. Because of the ready availability, low cost ($0.01/g), good surface area (30 M²/g) and ease of operation of a continuous cylindrical reactor, the high silica fabric was chosen. The immobilized enzyme had a pH optimum shifted to the alkaline side (from 6.5 to 7.5) and had a reduced temperature optimum (from 60°C to 50-55°C). Reuse efficiency showed 65% reduction in the overall activity of the immobilized enzyme after 10 cycles of 48 h each. Continuous operation at 55°C of a cylindrical reactor of 141 ml capacity, using the immobilized enzyme (80 g of high - silica fabric containing 114 mg of purified enzyme) gave a maximum productivity of 10.2 g of cyclodextrins L^-1 h,^-1, at a dilution rate of 0.32 h^-1 and a substrate concentration of 20 g L^-1. The half life of the biocatalyst was found to be 22 days, which could be further improved by using a lower operating temperature. Over the useful life time of the immobilized biocatalyst (22 days), the total Cyclodextrin produced was of the order of 88 Kg.     

Optimisation of growth medium for the production of cyclodextrin glucanotransferase from Bacillus stearothermophilus HR1 using response surface methodology

Cyclodextrin glucanotransferase (CGTase) activity was observed when the bacterium was grown in the medium at various initial pH values, containing carbon, nitrogen, phosphorus and mineral salt sources at 50 °C for 24 h in the shake flasks. The optimisation of this growth medium was carried out using response surface methodology. The design contains a total of 32 experimental trials involving 10 star points and 6 replicates at the centre points. The design was employed by selecting sago starch, peptone from casein, K₂HPO₄, CaCl₂ and initial pH as five independent variables in this study. The optimal calculated values of tested variables for maximal production of CGTase were found to be comprised of: sago starch, 16.02 g/l; peptone from casein, 20 g/l; K₂HPO₄, 1.4 g/l; CaCl₂, 0.2 g/l and initial pH, 7.54 with a predicted CGTase activity of 14.20 U/ml. These predicted optimal parameters were tested in the laboratory and the final CGTase activity obtained was very close to the predicted value at 14.80 U/ml.     

Enzymatic preparation of fatty acid esters of sugar alcohols by condensation in acetone using a packed-bed reactor with immobilized Candida antarctica lipase

Mono- and dilauroyl arabitols, ribitols, xylitols and sorbitols were synthesized batchwise or continuously at 50°C or 60°C by condensation catalyzed by an immobilized Candida antarctica lipase in acetone. Continuous production was realized using a system where a column packed with sugar alcohol and a packed-bed reactor with the immobilized lipase were connected in series. The concentrations of the mono- and dilauroyl esters of each sugar alcohol became almost constant at mean residence times of 15 min or longer in the packed-bed reactor. The monolauroyl, monomyristoyl and monopalmytoyl arabitols, ribitols, xylitols and sorbitols were continuously produced using the reactor system at 60°C, and the productivity was in the range of 1.3–2.0 kg L^?1-reactor·day except for the fatty acid esters of sorbitol, the productivity of which was 0.6–0.8 kg L^?1-reactor·day.     

Enzymatic preparation of fatty acid esters of sugar alcohols by condensation in acetone using a packed-bed reactor with immobilized Candida antarctica lipase

Mono- and dilauroyl arabitols, ribitols, xylitols and sorbitols were synthesized batchwise or continuously at 50°C or 60°C by condensation catalyzed by an immobilized Candida antarctica lipase in acetone. Continuous production was realized using a system where a column packed with sugar alcohol and a packed-bed reactor with the immobilized lipase were connected in series. The concentrations of the mono- and dilauroyl esters of each sugar alcohol became almost constant at mean residence times of 15 min or longer in the packed-bed reactor. The monolauroyl, monomyristoyl and monopalmytoyl arabitols, ribitols, xylitols and sorbitols were continuously produced using the reactor system at 60°C, and the productivity was in the range of 1.3-2.0 kg L^-1-reactor·day except for the fatty acid esters of sorbitol, the productivity of which was 0.6-0.8 kg L^-1-reactor·day.