Cyclodextrin glucanotransferase immobilization onto functionalized magnetic double mesoporous core-shell silica nanospheres

Background Cyclodextrin glucanotransferase (CGTase) from Amphibacillus sp. NPST-10 was covalently immobilized onto amino-functionalized magnetic double mesoporous core-shell silica nanospheres (mag@d-SiO2@m-SiO2-NH2), and the properties of the immobilized enzyme were investigated. The synthesis process of the nanospheres included preparing core magnetic magnetite (Fe3O4) nanoparticles, coating the Fe3O4 with a dense silica layer, followed by further coating with functionalized or non-functionalized mesoporous silica shell. The structure of the synthesized nanospheres was characterized using TEM, XRD, and FT-IR analyses. CGTase was immobilized onto the functionalized and non-functionalized nanospheres by covalent attachment and physical adsorption. Results The results indicated that the enzyme immobilization by covalent attachment onto the activated mag@d-SiO2@m-SiO2-NH2, prepared using anionic surfactant, showed highest immobilization yield (98.1%), loading efficiency (96.2%), and loading capacity 58 µg protein [CGTase]/mg [nanoparticles]) which were among the highest yields reported so far for CGTase. Compared with the free enzyme, the immobilized CGTase demonstrated a shift in the optimal temperature from 50°C to 50-55°C, and showed a significant enhancement in the enzyme thermal stability. The optimum pH values for the activity of the free and immobilized CGTase were pH 8 and pH 8.5, respectively, and there was a significant improvement in pH stability of the immobilized enzyme. Moreover, the immobilized CGTase exhibited good operational stability, retaining 56% of the initial activity after reutilizations of ten successive cycles. Conclusion The enhancement of CGTase properties upon immobilization suggested that the applied nano-structured carriers and immobilization protocol are promising approach for industrial bioprocess for production of cyclodextrins using immobilized CGTase.

A new low molecular mass alkaline cyclodextrin glucanotransferase from Amphibacillus sp. NRC-WN isolated from an Egyptian soda lake

Background: Cyclodextrin glucanotransferase (CGTase) is one of the most industrially important enzymes used in the commercial production of cyclodextrins (CDs). Alkaliphilic bacteria have attracted much interest in the last few decades because of their ability to produce extracellular enzymes that are active and stable at high pH values. Here, we report the isolation of a new CGTase from alkaliphilic bacteria collected from Egyptian soda lakes and describe the purification and biochemical characterization of this CGTase. Results: Screening for CGTase-producing alkaliphilic bacteria from sediment and water samples collected from Egyptian soda lakes located in the Wadi Natrun valley resulted in the isolation of a potent CGTase-producing alkaliphilic bacterial strain, designated NRC-WN. Strain NRC-WN was belonging to genus Amplibacullus by 16S rDNA sequence analysis (similarity: ca. 98%). Among the tested nitrogen and carbon sources, peptone (0.15%, w/v) and soluble starch (0.4%, w/v) allowed maximal CGTase production by Amphibacillus sp. NRC-WN. CGTase was successfully purified from Amphibacillus sp. NRC-WN up to 159.7-fold through a combination of starch adsorption and anion exchange chromatography, resulting in a yield of 84.7%. SDS-PAGE analysis indicated that the enzyme was purified to homogeneity and revealed an estimated molecular mass of 36 kDa, which makes it one of the smallest CGTases reported in the literature. The purified enzyme exhibited maximum activity at 50ºC and was stable up to 70ºC, retaining 93% of its initial activity after treatment for 1 hr. Furthermore, Ca2+ ions (10 mM) significantly enhanced the thermal stability of the CGTase. The purified enzyme was active and stable over a wide pH range, showing maximal activity at pH 9.5. The enzyme was significantly stimulated by Zn2+, Ca2+ and Co2+ but was completely inhibited in the presence of Fe3+ and mercaptoethanol. The Km and Vmax values of the purified CGTase were estimated to be 0.0434 mg/ml and 3,333.3 mg β-CD/ml/min, respectively. β-CD was the predominant product of starch degradation by the Amphibacillus sp. NRC-WN CGTase, followed by α-and γ-CDs. Conclusions: A new low molecular mass alkaline CGTase was purified from a newly identified alkaliphilic Amphibacillus sp. NRC-WN isolate from the Egyptian soda lakes. The enzyme showed promising thermal and pH stability and a high affinity toward starch as a natural substrate.

Immobilization of cyclodextrin glucanotransferase on aminopropyl-functionalized silica-coated superparamagnetic nanoparticles

Background: Cyclodextrin glycosyltransferase (CGTase) from Amphibacillus sp. NPST-10 was successfully covalently immobilized on aminopropyl-functionalized silica coated superparamagnetic nanoparticles; and the properties of immobilized enzyme were investigated. The synthesis process included preparing of core magnetic magnetite (Fe3O4) nanoparticles using solvothermal synthesis; followed by coating of Fe3O4 nanoparticles with dense amino-functionalized silica (NH2-SiO2) layer using in situ functionalization method. The structure of synthesized Fe3O4@NH2-SiO2 nanoparticles was characterized using TEM, XRD, and FT-IR analysis. Fe3O4@NH2-SiO2 nanoparticles were further activated by gluteraaldehyde as bifunctional cross linker, and the activated nanoparticles were used for CGTase immobilization by covalent attachment. Results: Magnetite nanoparticles was successfully synthesized and coated with and amino functionalized silica layer (Fe3O4/NH2-SiO2), with particle size of 50-70 nm. The silica coated magnetite nanoparticles showed with saturation magnetization of 65 emug-1, and can be quickly recovered from the bulk solution using an external magnet within 10 sec. The activated support was effective for CGTase immobilization, which was confirmed by comparison of FT-IR spectra of free and immobilized enzyme. The applied approach for support preparation, activation, and optimization of immobilization conditions, led to high yields of CGTase immobilization (92.3%), activity recovery (73%), and loading efficiency (95.2%); which is one of the highest so far reported for CGTase. Immobilized enzyme showed shift in the optimal temperature from 50 to 55ºC, and significant enhancement in the thermal stability compared with free enzyme. The optimum pH for enzyme activity was pH 8 and pH 7.5 for free and immobilized CGTase, respectively, with slight improvement of pH stability of immobilized enzyme. Furthermore, kinetic studies revealed that immobilized CGTase had higher affinity toward substrate; with k m values of 1.18 ± 0.05 mg/ml and 1.75 ± 0.07 mg/ml for immobilized and free CGTase, respectively. Immobilized CGTase retained 87% and 67 of its initial activity after 5 and 10 repeated batches reaction, indicating that immobilized CGTase on Fe3O4/NH2-SiO2 had good durability and magnetic recovery. Conclusion: The improvement in kinetic and stability parameters of immobilized CGTase makes the proposed method a suitable candidate for industrial applications of CGTase. To best of our knowledge, this is the first report about CGTase immobilization on silica coated magnetite nanoparticles.

Effects of substrates and reaction conditions on production of cyclodextrins using cyclodextrin glucanotransferase from newly isolated Bacillus agaradhaerens KSU-A11

The effects of reaction conditions on cyclodextrins (CDs) production by CGTase from newly isolated Bacillus agaradhaerens KSU-A11 is reported. Among six types of starch tested, potato starch gave highest starch conversion into CDs. In addition, CDs yield was about three fold higher when using gelatinized potato starch in comparison to raw starch. The total CDs production was increased with increasing pH, showing maximum starch conversion at pH 10. Furthermore, the proportion of gamma-CD was relatively higher under slightly acidic-neutral conditions than at alkaline pH with a maximum proportion of 35.6 percent at pH 7 compared to 7.6 percent at pH 10. Maximum starch conversion into CDs was seen at reaction temperature of 55ºC. Lower reaction temperature led to higher proportion of gamma-CD with maximum percentage at 35ºC. Cyclization reaction was significantly promoted in the presence CaCl2 (10 mM), while in the presence of ethyl alcohol there was significant decrease in CD production particularly at high concentration. beta-CD was the major product up to 1 hr reaction period with traces of alpha-CD and no detectable gamma-CD. However, as the reaction proceed, gamma-CD started to be synthesised and alpha-CD concentration increased up to 4 hrs, where the CDs ratios were 0.27:0.65:0.07 for alpha-CD:beta-CD:gamma-CD, respectively. In addition, optimum CGTase/starch ratio was obtained at 80 U/g starch, showing highest starch conversion into CDs. All the parameters involved have been shown to affect the products yield and/or specificity of B. agaradhaerens KSU-A11 CGTase.

Isolation and characterization of novel potent Cr(VI) reducing alkaliphilic amphibacillus sp. ksuCr3 from hypersaline soda lakes

A strain KSUCr3 with extremely high Cr(VI)-reducing ability under alkaline conditions was isolated from hypersaline soda lakes and identified as Amphibacillus sp. on the basis of 16S rRNA gene sequence analysis. The results showed that Amphibacillus sp. strain KSUCr3 was tolerance to very high Cr(VI) concentration (75 mM) in addition to high tolerance to other heavy metals including Ni2+ (100 mM), Mo2+ (75 mM), Co2+ (5 mM), Mn2+ (100 mM), Zn2+ (2 mM), Cu2+ (2 mM) and Pb (75 mM). Strain KSUCr3 was shown to be of a high efficiency in detoxifying chromate, as it could rapidly reduce 5 mM of Cr(VI) to a non detectable level over 24 hrs. In addition, strain KSUCr3 could reduce Cr(VI) efficiently over a wide range of initial Cr(VI) concentrations (1-10 mM) in alkaline medium under aerobic conditions without significant effect on the bacterial growth. Addition of glucose, NaCl and Na2CO3 to the culture medium caused a dramatic increase in Cr(VI)-reduction by Amphibacillus sp. strain KSUCr3. The maximum chromate removal was exhibited in alkaline medium containing 1.5 percent Na2CO3, 0.8 percent glucose, and 1.2 percent NaCl, at incubation temperature of 40ºC and shaking of 100 rpm. Under optimum Cr(VI) reduction conditions, Cr(VI) reduction rate reached 237 uMh¹ which is one of the highest Cr(VI) reduction rate, under alkaline conditions and high salt concentration, compared to other microorganisms that has been reported so far. Furthermore, the presence of other metals, such as Ni2+, Co2+, Cu2+ and Mn2+ slightly stimulated Cr(VI)-reduction ability by the strain KSUCr3.The isolate, Amphibacillus sp. strain KSUCr3, exhibited an ability to repeatedly reduce hexavalent chromium without any amendment of nutrients, suggesting its potential application in continuous bioremediation of Cr(VI). The results also revealed the possible isolation of potent heavy metals resistant bacteria from extreme environment such as hypersaline soda lakes.