Cyclomaltodextrin Glucanotransferase Research Articles

USO DE AGENTES COMPLEXANTES PARA O AUMENTO NA PRODUÇÃO DIRECIONADA DE CICLODEXTRINAS POR ENZIMA COMERCIAL TORUZYME®

USE OF COMPLEXING AGENTS FOR INCREASED TARGETED PRODUCTION OF CYCLODEXTRINS BY COMMERCIAL ENZYME TORUZYME®. Cyclodextrins (CDs) are cyclic oligosaccharides, products of the intramolecular transglycosylation reaction promoted by the enzyme cyclomaltodextrin glucanotransferase (CGTase). The most commonly produced CDs are a-CD, b-CD and g-CD, composed of 6, 7 and 8 glucose molecules, respectively. They form non-covalent inclusion complexes with several molecules, increasing their stability and/or solubility. Due to the importance of CDs and the various industrial applications, this work aimed to use the commercial enzyme Toruzyme® to obtain CDs and direct its production to a-CD or b-CD through the use of ethanol and complexing agents in the reaction medium. The reaction medium added with ethanol and cyclohexanecarboxylic acid directed the production to b-CD, reaching 47.09 mg mL-1 of this molecule. The production medium containing ethanol and stearic acid proved to be more advantageous for the production of a-CD and, after 48 h of reaction, the production of a-CD reached 19.57 mg mL-1, a value six times greater than that of amount of b-CD produced. With this study it was possible to prove that with the use of ethanol and complexing agents in the reaction medium it is possible to direct the production of a certain CD and, thus, make different CGTases economically viable for industrial use.

RNAseq analysis of Cellvibrio japonicus during starch utilization differentiates between genes encoding carbohydrate active enzymes controlled by substrate detection or growth rate.

Understanding the bacterial metabolism of starch is important as this polysaccharide is a ubiquitous ingredient in foods, supplements, and medicines, all of which influence gut microbiome composition and health. Our RNAseq and growth data set provides a valuable resource to those who want to better understand the regulation of starch utilization in Gram-negative bacteria. These data are also useful as they provide an example of how to approach studying a starch-utilizing bacterium that has many putative amylases by coupling transcriptomic data with growth assays to overcome the potential challenges of functional redundancy. The RNAseq data can also be used as a part of larger meta-analyses to compare how C. japonicus regulates carbohydrate active enzymes, or how this bacterium compares to gut microbiome constituents in terms of starch utilization potential.

Synthesis of isomaltooligosaccharides using 4-O-α-d-isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase.

Isomaltooligosaccharides (IMOs), including isomaltose, are valuable oligosaccharides, and the development of methods to synthesize high-purity IMOs has long been underway. We recently discovered a novel enzyme, 4-O-α-d-isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase (IMM-4IH), that showed promise for improving the synthesis process. In this study, we establish methods for synthesizing isomaltose and IMOs consisting of a variety of degrees of polymerization from starch using IMM-4IH. With 5% substrate, by combining IMM-4IH with 1,4-α-glucan 6-α-glucosyltransferase from Bacillus globisporus N75, the yield of isomaltose was 63.0%; incorporating isoamylase and cyclomaltodextrin glucanotransferase increased the yield to 75.3%. On the other hand, by combining IMM-4IH with 1,4-α-glucan 6-α-glucosyltransferase from Paenibacillus sp. PP710, IMOs were synthesized. The inclusion of isoamylase and α-amylase led to the 136 mM IMOs, consisting of oligosaccharides from isomaltose to isomaltodecaose, from 10% starch. The development of these efficient methods will be an important contribution to the industrial production of IMOs.

A multi-enzymatic cascade reaction for the synthesis of bioactive C-oligosaccharides

C-Oligosaccharides are rare in nature and possess diverse bioactivities. However, their chemical synthesis faces many challenges. In this work, enzymatic introduction of C-linked sugar chains to target aglycones was successfully achieved by multi-enzymatic cascade reactions. A C-glycosyltransferase from Aloe barbadensis was employed to introduce the first C-linked glucose and then a cyclomaltodextrin glucanotransferase from Bacillus licheniformis was used to extend the sugar chain. A total of twenty C-oligosaccharides with 2–6 sugars were synthesized from scale-up reactions and exhibited good water solubility and sodium-dependent glucose transporter 2 (SGLT2) inhibitory activity. Furthermore, a glucoamylase was used to control the length of the sugar chain and the C-maltosides were efficiently synthesized. These findings not only expanded the structural diversity of C-oligosaccharides, but also provided a strategy for the modification of C-glycoside drugs to improve the druggability.

Binding Specificity of a Novel Cyclo/Maltodextrin-Binding Protein and Its Role in the Cyclodextrin ABC Importer System from Thermoanaerobacterales.

Extracellular synthesis of functional cyclodextrins (CDs) as intermediates of starch assimilation is a convenient microbial adaptation to sequester substrates, increase the half-life of the carbon source, carry bioactive compounds, and alleviate chemical toxicity through the formation of CD-guest complexes. Bacteria encoding the four steps of the carbohydrate metabolism pathway via cyclodextrins (CM-CD) actively internalize CDs across the microbial membrane via a putative type I ATP-dependent ABC sugar importer system, MdxEFG-(X/MsmX). While the first step of the CM-CD pathway encompasses extracellular starch-active cyclomaltodextrin glucanotransferases (CGTases) to synthesize linear dextrins and CDs, it is the ABC importer system in the second step that is the critical factor in determining which molecules from the CGTase activity will be internalized by the cell. Here, structure-function relationship studies of the cyclo⁄maltodextrin-binding protein MdxE of the MdxEFG-MsmX importer system from Thermoanaerobacter mathranii subsp. mathranii A3 are presented. Calorimetric and fluorescence studies of recombinant MdxE using linear dextrins and CDs showed that although MdxE binds linear dextrins and CDs with high affinity, the open-to-closed conformational change is solely observed after α- and β-CD binding, suggesting that the CM-CD pathway from Thermoanaerobacterales is exclusive for cellular internalization of these molecules. Structural analysis of MdxE coupled with docking simulations showed an overall architecture typically found in sugar-binding proteins (SBPs) that comprised two N- and C-domains linked by three small hinge regions, including the conserved aromatic triad Tyr193/Trp269/Trp378 in the C-domain and Phe87 in the N-domain involved in CD recognition and stabilization. Structural bioinformatic analysis of the entire MdxFG-MsmX importer system provided further insights into the binding, internalization, and delivery mechanisms of CDs. Hence, while the MdxE-CD complex couples to the permease subunits MdxFG to deliver the CD into the transmembrane channel, the dimerization of the cytoplasmatic promiscuous ATPase MsmX triggers active transport into the cytoplasm. This research provides the first results on a novel thermofunctional SBP and its role in the internalization of CDs in extremely thermophilic bacteria.

The secretome of Thermococcus barophilus in the presence of carbohydrates and the potential role of the TrmBL4 regulator.

Global transcriptional regulators are crucial for supporting rapid adaptive responses in changing environments. In Thermococcales, the TrmB sugar-sensing regulator family is well represented but knowledge of the functional role/s of each of its members is limited. In this study, we examined the link between TrmBL4 and the degree of protein secretion in different sugar environments in the hyperthermophilic Archaeon Thermococcus barophilus. Although the absence of TrmBL4 did not induce any growth defects, proteomics analysis revealed different secretomes depending on the sugar and/or genetic contexts. Notably, 33 secreted proteins present in the supernatant were differentially detected. Some of these proteins are involved in sugar assimilation and transport, such as the protein encoded by TERMP_01455 (cyclomaltodextrin glucanotransferase), whereas others have intracellular functions, such as the protein encoded by TERMP_01556 (pyruvate: ferredoxin oxidoreductase Δsubunit). Then, using reverse transcription quantitative polymerase chain reaction experiments, we observed effective transcription regulation by TrmBL4 of the genes encoding at least two ABC-type transporters according to sugar availability.

Safety evaluation of the food enzyme cyclomaltodextrin glucanotransferase from the non-genetically modified Anoxybacillus caldiproteolyticus strain TCM3-539.

The food enzyme cyclomaltodextrin glucanotransferase ((1-4)-α-d-glucan:(1-4)-α-d-glucan 4-α-d-[(1-4)-α-d-glucano]-transferase; EC 2.4.1.19) is produced with the non-genetically modified bacteria Anoxybacillus caldiproteolyticus strain TCM3-539 by Hayashibara Co., Ltd. It is free from viable cells of the production strain. The food enzyme is intended to be used for the manufacture of glucosyl hesperidin and ascorbic acid 2-glucoside. Since residual amounts of total organic solids are removed by filtration, adsorption, chromatography and crystallisation, dietary exposure estimation was considered not necessary. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and one match with a respiratory allergen was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that the food enzyme does not give rise to safety concerns under the intended conditions of use.

Challenges and alternatives for the production of cyclodextrins from the CGTase enzyme from recombinant Bacillus subtilis WB800

Cyclodextrins (CDs) have the ability to encapsulate numerous molecules and have applicability in several industrial areas, however, their cost has made their use difficult. To seek alternatives that may enable the use of DCs, the present study evaluated the efficiency of the ultrafiltration process in a continuous system to produce CDs from the enzyme cyclomaltodextrin glucanotransferase (CGTase) from recombinant Bacillus subtilis WB800. The possibility of using the crude enzyme as an alternative means of producing CDs was also evaluated. All strategies evaluated in this research proved to be promising for the production of CDs, with the production of β-CD being the most efficient (average of 15 mmol/L) using crude recombinant enzyme and a temperature of 50 °C. Therefore, the results obtained can contribute to the reduction of stages and costs of production of CDs, favoring their industrial application.

Pudding Proteomics: Cyclomaltodextrin Glucanotransferase and Microbial Proteases Can Liquefy Extended Shelf Life Dairy Products.

In recent years, a lack of stability of dairy products with extended shelf life (e.g., yoghurt products, UHT desserts) has occurred, with the corresponding products liquefying significantly after days or weeks. This project aimed to identify the enzymes responsible for the liquefaction of the affected products based on differential proteomic analyses. No evidence was found for the presence of starch-degrading bacteria in the affected products. With zymography and proteome analysis, we detected the cause of liquefaction in a pudding by contamination of its aroma component with an engineered amylolytic enzyme, cyclomaltodextrin glucanotransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes. In addition, we detected contamination with Pseudomonas-derived proteolytic ATP-dependent Clp protease in one pudding batch and proteases in technically used amylases, which degraded β-caseins in another batch. Identification of these agents with liquefying properties in dairy products are useful for adjustment of production protocols and/or composition of additives, and thus shelf life extension.

Safety evaluation of glucosylated steviol glycosides as a food additive in different food categories.

The EFSA Panel on Food Additive and Flavourings (FAF) assessed the safety of glucosylated steviol glycosides proposed for use as a new food additive in different food categories. Glucosylated steviol glycosides consist of a mixture of glucosylated steviol glycosides, containing 1–20 additional glucose units bound to the parent steviol glycosides. Glucosylated steviol glycosides consist of not less than 95% (on dry, dextrin‐free, basis) of total steviol glycosides, comprised of glucosylated and parent steviol glycosides. Glucosylated steviol glycosides are produced via enzymatic bioconversion using cyclomaltodextrin glucanotransferase (CGTase) (EC 2.4.1.19), derived from a non‐genetically modified strain of Anoxybacillus caldiproteolyticus, that catalyses the transfer of glucose from starch to steviol glycosides mixtures isolated from the dried leaves of Stevia Rebaudiana. The Panel considered that the metabolism of glucosylated steviol glycosides is sufficiently similar to the already authorised steviol glycosides, and thus, the toxicological data previously assessed by the ANS Panel for steviol glycosides (E 960) were considered to support their safety as food additive. The existing acceptable daily intake (ADI) for steviol glycosides (E 960) of 4 mg/kg body weight (bw) per day expressed as steviol can also be applied to glucosylated steviol glycosides. The Panel concluded that there is no safety concern for the use of glucosylated steviol glycosides as a new food additive at the proposed use and use levels. The Panel recommended some modifications to the specifications proposed by the applicant for glucosylated steviol glycosides with respect to the assay, the definition of the proposed new food additive and the proposed maximum limits for arsenic.

Mining for novel cyclomaltodextrin glucanotransferases unravels the carbohydrate metabolism pathway via cyclodextrins in Thermoanaerobacterales

Carbohydrate metabolism via cyclodextrins (CM-CD) is an uncommon starch-converting pathway that thoroughly depends on extracellular cyclomaltodextrin glucanotransferases (CGTases) to transform the surrounding starch substrate to α-(1,4)-linked oligosaccharides and cyclodextrins (CDs). The CM-CD pathway has emerged as a convenient microbial adaptation to thrive under extreme temperatures, as CDs are functional amphipathic toroids with higher heat-resistant values than linear dextrins. Nevertheless, although the CM-CD pathway has been described in a few mesophilic bacteria and archaea, it remains obscure in extremely thermophilic prokaryotes (Topt ≥ 70 °C). Here, a new monophyletic group of CGTases with an exceptional three-domain ABC architecture was detected by (meta)genome mining of extremely thermophilic Thermoanaerobacterales living in a wide variety of hot starch-poor environments on Earth. Functional studies of a representative member, CldA, showed a maximum activity in a thermoacidophilic range (pH 4.0 and 80 °C) with remarkable product diversification that yielded a mixture of α:β:γ-CDs (34:62:4) from soluble starch, as well as G3–G7 linear dextrins and fermentable sugars as the primary products. Together, comparative genomics and predictive functional analysis, combined with data of the functionally characterized key proteins of the gene clusters encoding CGTases, revealed the CM-CD pathway in Thermoanaerobacterales and showed that it is involved in the synthesis, transportation, degradation, and metabolic assimilation of CDs.

Safety evaluation of the food enzyme cyclomaltodextrin glucanotransferase from Anoxybacillus caldiproteolyticus strain St-88.

The food enzyme cyclomaltodextrin glucanotransferase ((1→4)‐α‐d‐glucan 4‐α‐d‐[(1→4)‐α‐d‐glucano]‐transferase (cyclising), EC 2.4.1.19) is produced with Anoxybacillus caldiproteolyticus strain St‐88 by PureCircle USA. It is intended to be used in the manufacture of glycosylated steviol glycosides. Residual amounts of total organic solids are removed by the purification steps applied during the production of the modified steviol glycosides; consequently, dietary exposure was not calculated. For the same reason, toxicological studies other than assessment of allergenicity were not considered necessary. Similarity of the amino acid sequence of the food enzyme to those of known allergens was searched and four matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Efficient production of isomaltose and isomaltooligosaccharides from starch using 1,4-α-glucan 6-α-glucosyltransferase and isopullulanase.

We attempted to develop an efficient method for producing isomaltose, a disaccharide consisting of an α-(1→6)-linkage, from starch by combining enzymes of known activity. We found that the combination of 1,4-α-glucan 6-α-glucosyltransferase from Bacillus globisporus N75 and isopullulanase from Aspergillus brasiliensis ATCC 9642 led to the efficient synthesis of isomaltose. Inclusion of isoamylase and cyclomaltodextrin glucanotransferase resulted in increased efficiency, with production yields exceeding 70%. Furthermore, we considered that isomaltooligosaccharides could be synthesized from starch by combining 1,4-α-glucan 6-α-glucosyltransferase from Paenibacillus sp. PP710 and isopullulanase. In reactions that additionally utilized isoamylase and α-amylase, the total concentration of product, which included a series of isomaltooligosaccharides from isomaltose to isomaltodecaose, was 131 m m, and the ratio of 6-linked glucopyranosyl bonds to all bonds was 91.7% at a substrate concentration of 10%. The development of these manufacturing methods will accelerate the industrial production of isomaltose and isomaltooligosaccharides.

Safety evaluation of the food enzyme cyclomaltodextrin glucanotransferase from Paenibacillus illinoisenis strain 107.

The food enzyme cyclomaltodextrin glucanotransferase ((1→4)-α-d-glucan:(1→4)-α-d-glucan 4-α-d-[(1→4)-α-d-glucano]-transferase; EC 2.4.1.19) is produced with the non-genetically modified Paenibacillus illinoisenis strain 107 by Hayashibara Co., Ltd. The cyclomaltodextrin glucanotransferase food enzyme is intended to be used solely by the applicant in-house in starch processing for trehalose production. Since residual amounts of total organic solids (TOS) are removed by the purification steps applied during the production of trehalose, dietary exposure was not calculated. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panelidentified a no-observed-adverse-effect level (NOAEL) of 39 mg TOS/kg body weight per day, the highest dose tested. Similarity of the amino acid sequence to those of known allergens was searched and one match was found. The Panelconsidered that, under the intended condition of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure cannot be excluded, but the likelihood for such reactions to occur is considered to be low. Based on the data provided, the Panelconcluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Economic model for obtaining cyclodextrins from commercial cgtase

A repetitive batch process was employed followed by membrane ultrafiltration system to produce low-cost cyclodextrins (CDs) using commercial enzymes Toruzyme® cyclomaltodextrin glucanotransferase (CGTase) and its kinetic parameters were determined. The ultrafiltration system enabled the removalof inhibitory products from the reaction medium, allowing the enzyme to be recovered for reuse. A 10 kDa membrane was used to separate the different CDs produced by the CGTase. The substrates evaluated were maltodextrin, corn starch and cassava starch at 5, 10 and 15% (w/V), in the presence and absence of 10% (V/V) ethanol. After reaction for 132 h, 10% (w/V) cassava starch in the presence of ethanol provided the best results with 32.1 mg/mL of β-CD. Maximum production occurred after 72 h of reaction, with a yield of 87.4% of β-CD and an α-CD, β-CD and γ-CD production ratio of 1:1:0.08 g, respectively. When eight repetitive batches of 72 h followed by ultrafiltration and crystallization of β-CD were performed, 2.1 g of precipitate was obtained with a purity of 67.6% β-CD. The supernatant from the crystallization process was lyophilized and resulted in 35.3% α-CD. The developed model can be used industrially for the production of low cost CDs from easily obtained raw material.

ENZYMATIC PRODUCTION OF β-CYCLODEXTRIN FROM JACKFRUIT SEEDS (Artocarpus intergrifolia L.)

Abstract β-Cyclodextrin (β-CD) is a compound of great application for pharmaceutical and food industries, and is generally produced by starchy substrates by cyclomaltodextrin glucanotransferase (CGTase) action. The objective of this study was to produce β-CD using an alternative source of starch, such as jackfruit seed (Artocarpus intergrifolia L.) bran (JSB) by a commercial CGTase. The highest productivity of β-CD (52.10 µM/h.g) was obtained from 10 g of JSB in 100 mL of citrate buffer (10 mM / pH 6.0), with 17 % (v/v) of ethanol and 1.34 U/g of CGTase, at 59 °C for 4 hours. These same conditions were applied to starches extracted from the JSB (SJSB) and ginger (SG) and also, potato starch (SP). The SJSB and SG performances were similar to SP, and resulted in productivities around 2.7 times higher in relation to JSB. Thus, it is possible to conclude that both JSB and SJSB are promising substrates for β-CD production.

Immobilization of commercial cyclomaltodextrin glucanotransferase into controlled pore silica by the anchorage method and covalent bonding

Abstract Cyclomaltodextrin glucanotransferase (CGTase) is used mainly for the industrial production of cyclodextrins (CDs). Their potential use and availability have played a decisive role in increasing demand for CDs. The aim of the present study was to immobilize the commercial CGTase (Toruzyme® 3.0L) into controlled pore silica by surface anchoring (SCGT-A) and covalent bonding (SCGT-CB) and to apply the immobilized enzymes in the production of CDs. The SCGT-A method proved to be efficient due to its short immobilization time and, it was possible to immobilize 89.63% of CGTase, in terms of activity. Commercial CGTase immobilized by SCGT-CB provided an immobilization yield of 48.44%, also in terms of activity. There was an increase in total CD production of 60.97% and 34.90% for SCGT-A and SCGT-CB, respectively, in a reaction medium with 10% ethanol, compared to the same media without ethanol. Higher CD production occured at 24 and 48 h for SCGT-A and SCGT-CB methods, respectively. The use of SCGT-A was more efficient than SCGT-CB and free enzyme (CGT-F) after 24 h of reaction. The increase production of CDs obtained by the SCGT-A method and the demonstrated continuous production of CDs with the SCGT-CB method make the use of immobilized CGTase viable for industrial applications.

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 1.33µmol 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 157.78µmol β-CD/min/mL, while the activity of the CGTase from the wild strain was 9.54µmol 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.

Purification and properties of beta-cyclomaltodextrin glucanotransferase from Bacillus flexus SV 1.

Cyclomaltodextrin glucanotransferase is a unique enzyme that degrades starch into cyclic oligosaccharides called cyclodextrins, which have numerous applications in various industries such as pharmaceutical, textile, agricultural, cosmetics etc. Due to its wide applications, microorganism producing one type of cyclodextrin is of interest as it simplifies the down streaming process of separating mixture of cyclodextrins. In the present study, β-CGTase was isolated from Bacillus flexus SV 1 and biochemically characterized. Enzyme was purified by starch adsorption followed by DEAE cellulose column chromatography which resulted in a fold purification of 6.1, with a yield of 44.07%. Molecular weight of the purified enzyme was found to be 96.68 kDa, enzyme was monomeric in nature with a Km and Vmax of 0.08976 μmol mL-1 and 585.1 μmol/ml/min, respectively. Optimum pH and temperature of the purified enzyme was found to be 8.0 and 60 °C. Ca2+ showed significant increase in enzyme activity. The inhibition of enzyme by EDTA indicates that CGTase is a metalloenzyme. CGTase produced majorly β-CD and was alkalotolarent and active at high temperatures which is a promising candidate for various industries such as textile, food, agriculture, and pharmaceuticals.

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.