A recombinant cyclodextrin glycosyltransferase cloned from Paenibacillus sp. strain RB01 showed improved catalytic activity in coupling reaction between cyclodextrins and disaccharides

Recombinant cyclodextrin glycosyltransferase (CGTase) was obtained by cloning the PCR gene fragment from thermotolerant Paenibacillus sp. strain RB01 screened from hot spring area in Thailand and cloned into the Escherichia coli expression vector. The nucleotide sequence was analyzed and aligned. Nucleotide sequence of the recombinant CGTase contained an open reading frame of 2139 bp encoding 713 amino acid residues. The recombinant required one-third of culture time and neutral pH to produce CGTase compared to wild type. CGTases from both wild type and transformant were purified in parallel by starch adsorption and DEAE cellulose column. Their biochemical properties such as molecular weight, optimum pH and temperature were quite similar. However, the recombinant enzyme showed improved catalytic activity in the coupling reaction between cyclodextrins (CDs) and some disaccharides. Among several sugars tested with excess βCD, cellobiose was the best substrate followed by leucrose. Very low activity was observed with trehalose, lactose and mellibiose. Sucrose and raffinose showed no activity. The K _m and other kinetic parameters of recombinant enzyme were determined for cellobiose and several cyclodextrin derivatives. Recombinant CGTase showed lower K _m for βCD and its derivatives, with improved activity compared to wild type enzyme.     

Purification and Refolding of Cyclodextrin Glycosyltransferase Expressed Escherichia coli

Recombinant DNA technology and protein engineering are currently utilized in the cost-effective production of pharmaceutical and industrial proteins with native conformation. Escherichia coli retains its dominant position as the first choice of host for speed, simplicity and well-established production protocols. However, protein production using recombinant E. coli occasionally encounters complex purification and refolding steps. This paper introduces an efficient scheme for purification andin vitro refolding of industrially important proteins including cyclodextrin glycosyltransferase (CGTase) expressed in recombinant E. coli employing a polycationic amino acid fusion system. Fusion of polycationic amino acids to CGTase allowed purification and refolding of CGTase to be simple and efficient. A novel CGTase production strategy will be discussed by considering recent progress in protein purification and refolding techniques.     

Effect of ethanol on the synthesis of large-ring cyclodextrins by cyclodextrin glucanotransferases

Cyclodextrin glucanotransferase (EC 2.4.1.19, CGTase) synthesizes cyclodextrins (CD) composed of 6 to more than hundred glucose units from amylose by an intramolecular transglycosylation reaction. The addition of ethanol to the reaction medium resulted in an increase of the yield of large-ring CD obtained with a CGTase from Bacillus sp. BT3-2 and Bacillus macerans. The presence of 15% ethanol in the reaction mixture with the CGTase from Bacillus sp. BT3-2 resulted in a 30% increase of the amounts of CD₁₀–CD₁₃ synthesized after 5 h of reaction. The addition of 20% ethanol increased the yield of CD₁₄–CD₂₁ up to 1000%. The hydrolysis of the large-ring CD by the CGTases was suppressed in the presence of ethanol. The ring-opening coupling cyclization reactions of the CGTase were effected differently by the organic solvent which may contribute to the observed increase of the yield and size of the CD obtained in the synthesis reactions.     

Synthesis of maltooligosyl fructofuranosides catalyzed by immobilized cyclodextrin glucosyltransferase using starch as donor

Cyclodextrin glucosyltransferase (CGTase) from Thermoanaerobacter sp. was covalently immobilized on Eupergit C and used for the synthesis of maltooligosyl fructofuranosides employing soluble starch as donor and sucrose as acceptor. Using a weight ratio starch-sucrose of 1:2, the conversion of starch into acceptor products catalyzed by soluble and immobilized CGTases was higher than 80% in 48 h. Under these conditions, the reaction was selective for the formation of maltosyl fructofuranoside.     

Effect of the reaction temperature on the transglycosylation reactions catalyzed by the cyclodextrin glucanotransferase from Bacillus macerans for the synthesis of large-ring cyclodextrins

The synthesis of cyclodextrins with from 6 to more than 50 glucose units by cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from Bacillus macerans was investigated. Analysis of the synthesized cyclic α-1,4-glucan products showed that a higher yield of large-ring cyclodextrins were obtained with a reaction temperature of 60 °C compared to 40 °C. The yield of large-ring cyclodextrins obtained at 60 °C represented about 50% of the total glucans employed in the reaction. Analysis of the cyclodextrin-forming cyclization reaction and of the coupling reaction of the CGTase resulting in the degradation of mainly the larger cyclic α-1,4-glucans indicated higher rates of the cyclization reaction at 60 °C compared to 40 °C while the opposite was found for the coupling reaction.     

In silico analysis of different signal peptides for the secretory production of recombinant human keratinocyte growth factor in Escherichia coli

BackgroundThe recombinant human truncated Keratinocyte growth factor (Palifermin) is the only FDA approved medicine for the treatment of oral mucositis. The Keratinocyte growth factor is a fairly unstable protein due to its high aggregation propensity and therefore its expression as a secretory protein may results in the production of a protein with more stability, higher solubility, better folding, enhanced biological activity, N-terminal authenticity and simplified downstream processing.ObjectiveThe aim of this study was in silico evaluation of 31 different secretory signal peptides to determine the best theoretical candidates for the secretory production of recombinant truncated human KGF in E. coli.MethodsThirty different prokaryotic signal peptides experimentally shown to be capable of recombinant protein secretion in E.coli, along with the native KGF signal peptide were selected for further investigations. The signal peptide sequences were retrieved from the UniProt database. The ability of SPs to act as a secretory leader peptide for rhKGF and the location of cleavage sites were predicted by SignalP 4.1. Physicochemical properties of the signal peptides, which may influence protein secretion, were analyzed by ProtParam and PROSOII. Furthermore, the mRNA secondary structure and Gibbs free energy profile of the selected SPs were analyzed in the fusion state with the rhKGF using Visual Gene Developer package.Results and ConclusionComputational analysis of the physicochemical properties affecting protein secretion identified Sec-B dependent OmpC, Bla, and StaI and SRP dependent TolB signal peptides as the best theoretical candidates for the secretory production of recombinant truncated human KGF in E.coli.     

Effect of organic solvents on the yield and specificity of cyclodextrins by recombinant cyclodextrin glucanotransferase (CGTase) from Anaerobranca gottschalkii

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is an enzyme that degrades starch and starch related glucans into cyclodextrins (CDs) by intramolecular transglycosylation reaction. The biochemical activity of recombinant CGTase from Anaerobranca gottschalkii for the yield and product specificity of cyclodextrins was investigated in the presence of organic solvents. Compared with the control of starch bioconversion, addition of various organic solvents generally increased the total CD and product specificity by affecting product inhibition and/or intermolecular transglycosylation reaction. The highest conversion (45 %) of starch to CDs was obtained in the presence of ethanol, while the simultaneous addition of two organic solvents, decanol-ethanol, comparatively showed a reduced total yield of 39 %. Despite this, the highest product ratio of 91 % α-CD, and 64 % β-CD was obtained in the presence of decanol and cyclohexane respectively. The effect of organic solvents on the yield and specificity of CD was attributed mainly to their effect on product inhibition and transglycosylation reaction. Although the use of two organic solvents showed almost a significant increase in total yield of CDs, it resulted in a comparatively lower specific product yield compared to their respective individual effect. Generally, normal enzyme activity was favoured at higher temperature of 65 °C, but the addition of organic solvents, in most cases, was found to decrease the bioconversion. Thus, the preferred optimal condition was reduced to 40 °C, where the maximal conversion of starch to CDs in general and α-CD in particular was achieved.     

Signal Peptide-Independent Secretory Expression and Characterization of Pullulanase from a Newly Isolated Klebsiella variicola SHN-1 in Escherichia coli

A strain with the power to produce extracellular pullulanase was obtained from the sample taken from a flour mill. By sequencing its 16S rDNA, the isolate was identified as Klebsiella variicola SHN-1. When the gene encoding pullulanase, containing the N-terminal signal sequence, was cloned into Escherichia coli BL21 (DE3), extracellular activity was detected up to 10 U/ml, a higher level compared with the results in published literature. Subsequently, the recombinant pullulanase was purified and characterized. The main end product from pullulan hydrolyzed by recombinant pullulanase was determined as maltotriose with HPLC, and hence, the recombinant pullulanase was identified as type I pullulanase, which could be efficiently employed in starch processing to produce maltotriose with higher purity and even to evaluate the purity of pullulan. To investigate the effect of signal peptide on secretion of the recombinant enzyme, the signal sequence was removed from the constructed vector. However, secretion of pullulanase in E. coli was not influenced, which was seldom reported previously. By localizing the distribution of pullulanase on subcellular fractions, the secretion of recombinant pullulanase in E. coli BL21 (DE3) was confirmed, even from the expression system of nonsecretory type without the assistance of signal peptide.     

Large‐Ring Cyclodextrins as Chiral Selectors for Enantiomeric Pharmaceuticals

Large‐ring cyclodextrins (CD) are cyclic glucans composed of 9 or more α‐1,4‐linked glucose units. They are minor side products of bacterial glucanotransferases (CGTases, EC 2.4.1.19) and have previously been available only in very small amounts for studies of their properties in supramolecular complex formation reactions. We engineered a CGTase to synthesize mainly large‐ring CD facilitating their preparation in larger amounts. By reversed phase chromatography, we obtained single CD samples composed of 10 to 12 glucose units (CD10, CD11, and CD12) with a purity of >90 %. Their identity was confirmed by high resolution mass spectrometry and fragmentation analysis. We demonstrated the non‐toxicity of CD10–CD12 for human cell lines by a cell proliferation assay and impedimetric monitoring. We then showed that CD10 and CD11 are efficient chiral selectors for the capillary electrophoretic separation of the enantiomeric pharmaceuticals fluvastatin, mefloquine, carvedilol, and primaquine.     

Purification and Properties of a New Thermostable Cyclodextrin Glucanotransferase from Bacillus pseudalcaliphilus 8SB

A new cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from an alkaliphilic halotolerant Bacillus pseudalcaliphilus 8SB was studied in respect to its γ-cyclizing activity. An efficient conversion of a raw corn starch into only two types of cyclodextrins (β- and γ-CD) was achieved by the purified enzyme. Crude enzyme obtained by ultrafiltration was purified up to fivefold by starch adsorption with a recovery of 62% activity. The enzyme was a monomer with a molecular mass 71 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. The CGTase exhibited two pH optima, at pH 6.0 and 8.0, and was at most active at 60 °C and pH 8.0. The enzyme retained more than 80% of its initial activity in a wide pH range, from 5.0 to 11.0. The CGTase was strongly inhibited by 15 mM Cu(2+), Fe(2+), Ag(+), and Zn(2+), while some metal ions, such as Ca(2+), Na(+), K(+), and Mo(7+), exerted a stimulating effect in concentration of 5 mM. The important feature of the studied CGTase was its high thermal stability: the enzyme retained almost 100% of its initial activity after 2 h of heating at 40-60 °C; its half-life was 2 h at 70 °C in the presence of 5 mM Ca(2+). The achieved 50.7% conversion of raw corn starch into 81.6% β- and 18.4% γ-CDs after 24 h enzyme reaction at 60 °C and pH 8.0 makes B. pseudalcaliphilus 8SB CGTase industrially important enzyme for cyclodextrin production.     

Synthesis of Large-Ring Cyclodextrins by Cyclodextrin Glucanotransferases from Bacterial Isolates

The synthesis of large-ring cyclodextrins (LR-CD) with cyclodextrin glucanotransferase (CGTase) preparations from the bacterial isolates BT3, BT4, BT25 and BT57 was investigated. All CGTase preparations catalyzed the synthesis of LR-CD with 5% soluble starch or 2% synthetic amylose as substrate. The amount and size of LR-CD synthesized depended on the reaction time and on the particular CGTase preparation used. The yield of LR-CD obtained with the enzyme preparation from BT3 and synthetic amylose as substrate was about 18% of the total glucan amount after a reaction time of 23 h.     

Tuning the Outcome of Enzyme-Mediated Dynamic Cyclodextrin Libraries to Enhance Template Effects

Enzyme-mediated dynamic combinatorial chemistry combines the concept of thermodynamically controlled covalent self-assembly with the inherent biological relevance of enzymatic transformations. A system of interconverting cyclodextrins has been explored, in which the glycosidic linkage is rendered dynamic by the action of cyclodextrin glucanotransferase (CGTase). External factors, such as pH, temperature, solvent, and salinity are reported to modulate the composition of the dynamic cyclodextrin library. Dynamic libraries of cyclodextrins (CDs) could be obtained in wide ranges of pH (5.0–9.0), temperature (5–37 °C), and salinity (up to 7.5 m NaNO₃), and with high organic solvent content (50 % by volume of ethanol), showing that enzyme-mediated dynamic systems can be robust and not limited to physiological conditions. Furthermore, it is demonstrated how strategic choice of reaction conditions can enhance template effects, in this case, to achieve highly selective production of α-CD, an otherwise challenging target due to competition from the structurally similar β-CD.     

Deep protein representations enable recombinant protein expression prediction

A crucial process in the production of industrial enzymes is recombinant gene expression, which aims to induce enzyme overexpression of the genes in a host microbe. Current approaches for securing overexpression rely on molecular tools such as adjusting the recombinant expression vector, adjusting cultivation conditions, or performing codon optimizations. However, such strategies are time-consuming, and an alternative strategy would be to select genes for better compatibility with the recombinant host. Several methods for predicting soluble expression are available; however, they are all optimized for the expression host Escherichia coli and do not consider the possibility of an expressed protein not being soluble. We show that these tools are not suited for predicting expression potential in the industrially important host Bacillus subtilis. Instead, we build a B. subtilis-specific machine learning model for expressibility prediction. Given millions of unlabelled proteins and a small labeled dataset, we can successfully train such a predictive model. The unlabeled proteins provide a performance boost relative to using amino acid frequencies of the labeled proteins as input. On average, we obtain a modest performance of 0.64 area-under-the-curve (AUC) and 0.2 Matthews correlation coefficient (MCC). However, we find that this is sufficient for the prioritization of expression candidates for high-throughput studies. Moreover, the predicted class probabilities are correlated with expression levels. A number of features related to protein expression, including base frequencies and solubility, are captured by the model.     

Cyclodextrins as Protective Agents of Protein Aggregation: An Overview

Cyclodextrins are extensively used in different fields (e.g., catalysis, chromatography, pharma, supramolecular chemistry, bioorganic chemistry, and bioinorganic chemistry), and their applications have been widely reviewed. Their main application in the field of pharmaceutical is as a drug carrier. This review overviews, for the first time, the use of cyclodextrins and their derivatives as antiaggregant agents in a number of proteins (e.g., amyloid‐β, insulin, recombinant human growth hormone, prion protein, transthyretin, and α‐synuclein) and some multimeric enzymes. There are many diseases that are correlated to protein misfolding and amyloid formation processes affecting numerous organs and tissues. There are over 30 different amyloid proteins and a number of corresponding diseases. Alzheimer's disease is the most common neurodegenerative disease. Treatment of these diseases is still a goal to reach, and many molecules are studied in this perspective. Cyclodextrins have also been studied, and they show great potential; as such, further studies could be very promising. This review aims to be a stimulus for the design of new cyclodextrin derivatives to obtain multifunctional systems with antiaggregant activity.     

Expression,Purification, and Characterization of Endo-β-N-Acetylglucosaminidase H Using Baculovirus-Mediated Silkworm Protein Expression System

Endo-β-N-acetylglucosaminidase (Endo H) from Streptomyces plicatus hydrolyzes the core di-GlcNAc units of asparagine-linked oligosaccharides and is regarded as an important tool for glycobiology research. In the present study, we established a large-scale system to produce secreted Endo H using a silkworm-baculovirus expression system (silkworm-BES). The recombinant Endo H purified from silkworm hemolymph had activity comparable to that from recombinant Escherichia coli. As well as its well-characterized substrate RNase B, the Endo H from silkworm-BES was able to deglycosylate the high-mannose glycoproteins from silkworm hemolymph. Interestingly, the secretion amount of recombinant Endo H was significantly varied among the different silkworm strains, which could provide valuable information for larger-scale protein productions from silkworm-BES.     

Production and purification of CGTase of alkalophylicBacillus isolated from Brazilian soil

Alkalophylic bacilli that produce cyclodextringlycosyltransferase (CGTase) were isolated from Brazilian soil, with a scheme of two plating steps. In the first step, the bacterial isolate forms a halo in the cultivation medium that contains γ-cyclodextrin (CD) complexing dyes. The CGTase of an isolate was purified 157-fold by biospecific affinity chromatography, with β-CD showing a mol wt of 77,580 Daltons. It produces a γ- to β-CD ratio of 0.156 and a small amount of α-CD, using maltodextrin 10% as substrate, at 50°C, pH 8.0 and 22 h reaction time, reaching 21.4% conversion of the substrate to cyclodextrins. In the second screening step, the isolates chosen give larger halos with β-CD complexing dyes, and smaller halos with β-CD complexing dyes, leading to a 30% improvement in γ-CD selectivity, although at lower total yield for cyclodextrins (11.5%).     

Gamma-Cyclodextrin Production Using Cyclodextrin Glycosyltransferase from Bacillus clarkii 7364

The production of cyclodextrins (CDs) by cyclodextrin glycosyltransferase (CGTase) from Bacillus clarkii 7364 was studied. Forty-seven percent (w/w) conversion rate to ??-CD was obtained in the process performed by reacting 5 U per gram of starch CGTase with 15?% (w/v) soluble starch in 0.025?M sodium phosphate?CNaOH buffer (pH 12) at 55?°C in the presence of 2?% (w/v) glycyrrhizic acid. Meanwhile, the ratio of ??:??-CD was 89:11, with negligible formation of ??-CD. Under these conditions, there is a significant increase in overall production of CDs and a marked change in product selectivity for ??-CD. The possible mechanisms were discussed upon different product profiles with respect to the size and amount of CDs synthesized at different reaction conditions. The approach described here can be easily applied to an enzymatic process for the production of ??-CD on an industrial scale, and such high selectivity, at high conversions, is especially attractive from a commercial perspective.     

Evaluation of solid and submerged fermentations for the production of cyclodextrin glycosyltransferase by Paenibacillus campinasensis H69-3 and characterization of crude enzyme

Cyclodextrin glycosyltransferase (CGTase) is an enzyme that produces cyclodextrins from starch by an intramolecular transglycosylation reaction. Cyclodextrins have been shown to have a number of applications in the food, cosmetic, pharmaceutical, and chemical industries. In the current study, the production of CGTase by Paenibacillus campinasensis strain H69-3 was examined in submerged and solid-state fermentations. P. campinasensis strain H69-3 was isolated from the soil, which grows at 45°C, and is a Gramvariable bacterium. Different substrate sources such as wheat bran, soybean bran, soybean extract, cassava solid residue, cassava starch, corn starch, and other combinations were used in the enzyme production. CGTase activity was highest in submerged fermentations with the greatest production observed at 48–72 h. The physical and chemical properties of CGTase were determined from the crude enzyme produced from submerged fermentations. The optimum temperature was found to be 70–75°C, and the activity was stable at 55°C for 1 h. The enzyme displayed two optimum pH values, 5.5 and 9.0 and was found to be stable between a pH of 4.5 and 11.0.     

Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the <Emphasis Type="Italic">Aedes aegypti</Emphasis> Mosquito Utilizing <Emphasis Type="Italic">E. coli</Emphasis> as a host

Background

Studying proteins and enzymes involved in important biological processes in the Aedes aegypti mosquito is limited by the quantity that can be directly isolated from the mosquito. Adding to this difficulty, digestive enzymes (midgut proteases) involved in metabolizing blood meal proteins require a more oxidizing environment to allow proper folding of disulfide bonds. Therefore, recombinant techniques to express foreign proteins in Escherichia coli prove to be effective in producing milligram quantities of the expressed product. However, with the most commonly used strains having a reducing cytoplasm, soluble expression of recombinant proteases is hampered. Fortunately, new E. coli strains with a more oxidizing cytoplasm are now available to ensure proper folding of disulfide bonds.

Results

Utilizing an E. coli strain with a more oxidizing cytoplasm (SHuffle® T7, New England Biolabs) and changes in bacterial growth temperature has resulted in the soluble expression of the four most abundantly expressed Ae. aegypti midgut proteases (AaET, AaSPVI, AaSPVII, and AaLT). A previous attempt of solubly expressing the full-length zymogen forms of these proteases with the leader (signal) sequence and a modified pseudo propeptide with a heterologous enterokinase cleavage site led to insoluble recombinant protein expression. In combination with the more oxidizing cytoplasm, and changes in growth temperature, helped improve the solubility of the zymogen (no leader) native propeptide proteases in E. coli. Furthermore, the approach led to autocatalytic activation of the proteases during bacterial expression and observable BApNA activity. Different time-points after bacterial growth induction were tested to determine the time at which the inactive (zymogen) species is observed to transition to the active form. This helped with the purification and isolation of only the inactive zymogen forms using Nickel affinity.

Conclusions

The difficulty in solubly expressing recombinant proteases in E. coli is caused by the native reducing cytoplasm. However, with bacterial strains with a more oxidizing cytoplasm, recombinant soluble expression can be achieved, but only in concert with changes in bacterial growth temperature. The method described herein should provide a facile starting point to recombinantly expressing Ae. aegypti mosquito proteases or proteins dependent on disulfide bonds utilizing E. coli as a host.

     

Enhancement of selectivity for producing γ-cyclodextrin

The production of cyclodextrins (CDs) by cyclodextrin-glycosyl-transferase (CGTase) from Bacillus firmus was studied, with respect to the effect of the source of starch upon CD yield and on the selectivity for producing γ-CD. Cyclodextrin production tests were run for 24 h at 50°C, pH 8.0, and 1 mg/L of CGTase, and substrates were maltodextrin or the starches of rice, potato, cassava, and corn hydrolyzed up to D. E. 10. Cornstarch was the best substrate for producing γ-CD. Later, glycyrrhizin (2.5% [w/v]), which forms a stable complex with γ-CD, was added to the cornstarch reaction medium and increased the yield of γ-CD to about four times that produced with only maltodextrin, but the total yield of CDs remained practically unchanged. Therefore, the results showed that the studied CGTase is capable of giving relatively high yield of γ-CD in the presence of glycyrrhizin as complexant and cornstarch as substrate.