Date of Award

4-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemical Engineering

Department

Chemical Engineering

First Advisor

Sulaiman Al-Zuhair

Abstract

Cyclodextrins (CDs) and their derivatives have attracted significant attention in the pharmaceutical, food, and textile industries, which has led to an increased demand for their production. CDs are typically produced by the action of cyclodextrin glycosyltransferase (CGTase) on starch. Owing to the relatively high cost of enzymes, the economic feasibility of the entire process strongly depends on the effective retention and recycling of CGTase in the reaction system, while maintaining the enzyme’s activity and stability. Previous supports used for this purpose such as silica and hydrogels, have numerous drawbacks, including enzyme leaching, activity loss and significant mass transfer limitations. The aim of this dissertation was to improve performance of immobilized CGTase by using metal–organic frameworks (MOFs), possessing better properties than conventional supports, as immobilization support. CGTase was immobilized on different synthesized MOFs, namely MIL-101, Cu-BTC, using either surface, covalent attachment or entrapment and compared to conventional support, namely Zeolite Y as well as Graphene nano-particles (GNP). The use of a calcium-based two-dimensional MOF, namely Ca-TMA, and modified Cu-BTC using N.N- dimethylcyclohexylamine to produce hierarchical H-Cu-BTC were also tested for enhanced enzyme capacity and reduced diffusional limitations of the large starch molecules. The adsorption capacity, the effect of immobilization on the secondary structures of CGTase and on the characterization of the support as well as the kinetic parameters of the free CGTase were assessed. The adsorption isotherms of CGTase on the tested MOFs were best represented by the Langmuir isotherm, with maximum adsorption capacities reaching 21, 30.6, 37.5, and 40 mg/g over Ca-TMA, Cu-BTC, microporous MIL-101 and GNP, respectively. The adsorption capacity was improved to 49.5 mg/g over H-Cu-BTC. These capacities were significantly higher than that observed using conventional Zeolite-Y, which did not exceed 6.1 mg/g, as well as other supports reported in previous literature. Characterization of the free supports using combination of X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Thermal Gravimetric Analysis (TGA) revealed that the structures of the MOFs remained intact post-CGTase immobilization. The deconvolution of the amide I band of the Fourier Transform Infrared spectra indicated that free CGTase molecules predominantly contain β-sheets (56% β-sheets, 38.5% α-helix and 5.5 % β-turns), with its composition changing over MIL-101 (84.1% β-sheets, no α-helix and 15.9 % β-turns), Ca-TMA (51.3% β-sheets, 37.5% α-helix and 11.2 % β-turns), and GNP (41.9% β-sheets, 18,1% α-helix and 40% β-turns. Lesser conformational changes were also observed using Cu-BTC (44% β-sheets, 37.5% α-helix and 11.2% β-turns) and H-Cu-BTC (76.1% β-sheets, 23.9% α-helix and no β-turns) supports. The immobilized CGTase on the different MOFs were tested for CDs production from starch, and the relative activity, reusability and mass-transfer limitations were investigated. The specific activity of the free CGTase used was 167 U/mg, which dropped upon immobilization to 28, 38, 65.2 and 98.5 U/mg protein on GNP, Ca-TMA, Cu-BTC, and H-CU-BTC, respectively. Reusability studies revealed that based on α-CD, MIL-101 showed 29% residual enzyme activity, which improved with covalent attachment via glutaraldehyde to 40%, Ca-TMA gave 33%, and GNP showed 74% relative activity after eight reaction cycles. Entrapment of CGTase within H-Cu-BTC led to residual CGTase activity of 87% after ten reaction cycles, compared to microporous Cu-BTC which gave 70% and presence of macropores and mesopores enhanced substrate mass transfer from 0.68 min-1 over microporous MOFs to 0.89 min-1 on macroporous H-Cu-BTC, thus, improved cyclodextrin production. This dissertation provides information on the effect of MOFs properties on immobilized CGTase performance, which can be used in developing robust CGTase-based biocatalysts for industrial application.

Arabic Abstract


اﻹﻧﺘﺎج اﻷﻧﺰﯾﻤﻲ ﻟﻠﺴﺎﯾﻜﻠﻮدﻛﺴﺘﺮﯾﻦ ﺑﺎﺳﺘﺨﺪام ﻧﺎﻗﻠﺔ اﻟﻐﻠﯿﻜﻮزﯾﻞ اﻟﺴﯿﻜﻠﻮدﯾﻜﺴﺘﺮﯾﻦ اﻟﻤﺜﺒﺖ ﻓﻲ اﻷطﺮ اﻟﻤﻌﺪﻧﯿﺔ اﻟﻌﻀﻮﯾﺔ (MOFs)

ﻟﻘﺪ اﺟﺘﺬﺑﺖ ﻣﺮﻛﺒﺎت اﻟﺴﯿﻜﻠﻮدﻛﺴﺘﺮﯾﻦ (CDs) وﻣﺸﺘﻘﺎﺗﮭﺎ اھﺘﻤﺎﻣًﺎ ﻛﺒﯿﺮً ا ﻓﻲ اﻟﺼﻨﺎﻋﺎت اﻟﺪواﺋﯿﺔ واﻟﻐﺬاﺋﯿﺔ واﻟﻨﺴﯿﺠﯿﺔ، ﻣﻤﺎ أدى إﻟﻰ زﯾﺎدة اﻟﻄﻠﺐ ﻋﻠﻰ إﻧﺘﺎﺟﮭﺎ. ﯾﺘﻢ إﻧﺘﺎج اﻟﺴﯿﻜﻠﻮدﻛﺴﺘﺮﯾﻦ ﻋﺎدة ﻋﻦ طﺮﯾﻖ ﻋﻤﻞ ﺳﯿﻜﻠﻮدﯾﻜﺴﺘﺮﯾﻦ ﺟﻠﯿﻜﻮزﯾﻞ ﺗﺮاﻧﺴﻔﯿﺮاز (CGTase) ﻋﻠﻰ اﻟﻨﺸﺎ. وﻧﻈﺮً ا ﻟﻠﺘﻜﻠﻔﺔ اﻟﻤﺮﺗﻔﻌﺔ ﻧﺴﺒﯿﺎً ﻟﻺﻧﺰﯾﻤﺎت، ﻓﺈن اﻟﺠﺪوى اﻻﻗﺘﺼﺎدﯾﺔ ﻟﻠﻌﻤﻠﯿﺔ ﺑﺮﻣﺘﮭﺎ ﺗﻌﺘﻤﺪ ﺑﺸﺪة ﻋﻠﻰ اﻻﺣﺘﻔﺎظ اﻟﻔﻌﺎل وإﻋﺎدة ﺗﺪوﯾﺮ CGTaseﻓﻲ ﻧﻈﺎم اﻟﺘﻔﺎﻋﻞ، ﻣﻊ اﻟﺤﻔﺎظ ﻋﻠﻰ ﻧﺸﺎط اﻹﻧﺰﯾﻢ واﺳﺘﻘﺮاره. اﻟﺪﻋﺎﻣﺎت اﻟﺴﺎﺑﻘﺔ اﻟﻤﺴﺘﺨﺪﻣﺔ ﻟﮭﺬا اﻟﻐﺮض ﻟﮭﺎ ﻋﯿﻮب ﻋﺪﯾﺪة، ﺑﻤﺎ ﻓﻲ ذﻟﻚ ﺗﺮﺷﯿﺢ اﻹﻧﺰﯾﻢ، وﻓﻘﺪان اﻟﻨﺸﺎط اﻷﻧﺰﯾﻤﻲ، وﻗﯿﻮد ﻛﺒﯿﺮة ﻋﻠﻰ ﻧﻘﻞ اﻟﻜﺘﻠﮫ. ﻛﺎن اﻟﮭﺪف ﻣﻦ ھﺬه اﻷطﺮوﺣﺔ ھﻮ ﺗﺤﺴﯿﻦ أداء CGTaseاﻟﻤﺜﺒﺖ ﻋﻦ طﺮﯾﻖ اﺳﺘﺨﺪام اﻷطﺮ اﻟﻤﻌﺪﻧﯿﺔ اﻟﻌﻀﻮﯾﺔ (MOFs)، اﻟﺘﻲ ﺗﻤﺘﻠﻚ ﺧﺼﺎﺋﺺ أﻓﻀﻞ ﻣﻦ اﻟﺪﻋﺎﻣﺎت اﻟﺘﻘﻠﯿﺪﯾﺔ، ﻛﺪﻋﺎﺋﻢ ﻟﻠﺘﺜﺒﯿﺖ. ﺗﻢ ﺗﺜﺒﯿﺖ CGTase ﻋﻠﻰ ﻣﺨﺘﻠﻒ ﻣﻦ إطﺎرات MOF اﻟﻤُﺼﻨﱠﻌﺔ، وھﻲ 101-MIL وCu-BTC، ﺑﺎﺳﺘﺨﺪام إﻣﺎ اﻟﺴﻄﺢ أو اﻟﻤﺮﻓﻖ اﻟﺘﺴﺎھﻤﻲ أو اﻻﻧﺤﺒﺎس، وﻣﻦ ﺛﻢ ﻣﻘﺎرﻧﺘﮫ ﺑﺎﻟﺪﻋﺎﻣﺔ اﻟﺘﻘﻠﯿﺪﯾﺔ، أي اﻟﺰﯾﻮﻟﯿﺖ Y وﻛﺬﻟﻚ ﺟﺰﯾﺌﺎت اﻟﺠﺮاﻓﯿﻦ اﻟﻨﺎﻧﻮﯾﺔ (GNP). ﺗﻢ أﯾﻀًﺎ اﺧﺘﺒﺎر اﺳﺘﺨﺪام MOF ﺛﻨﺎﺋﻲ اﻷﺑﻌﺎد اﻟﻘﺎﺋﻤﺔ ﻋﻠﻰ اﻟﻜﺎﻟﺴﯿﻮم، وھﻲ Ca-TMA، وCu-BTC اﻟﻤﻌﺪﻟﺔ ﺑﺎﺳﺘﺨﺪام ﺛﻨﺎﺋﻲ ﻣﯿﺜﯿﻞ ﺳﯿﻜﻠﻮھﯿﻜﺴﯿﻼﻣﯿﻦ ﻹﻧﺘﺎج H-Cu-BTC اﻟﮭﺮﻣﻲ ﻟﺘﻌﺰﯾﺰ ﻗﺪرة اﻹﻧﺰﯾﻢ وﺗﻘﻠﯿﻞ ﻗﯿﻮد اﻻﻧﺘﺸﺎر ﻟﺠﺰﯾﺌﺎت اﻟﻨﺸﺎ اﻟﻜﺒﯿﺮة. ﺗﻢ ﺗﻘﯿﯿﻢ ﻗﺪرة اﻻﻣﺘﺼﺎص وﺗﺄﺛﯿﺮ اﻟﺘﺜﺒﯿﺖ ﻋﻠﻰ اﻟﮭﯿﺎﻛﻞ اﻟﺜﺎﻧﻮﯾﺔ ﻟـ CGTase وﻋﻠﻰ ﺗﻮﺻﯿﻒ اﻟﺪﻋﻢ ﺑﺎﻹﺿﺎﻓﺔ إﻟﻰ اﻟﻘﯿﻢ اﻟﺤﺮﻛﯿﺔ ﻟـ CGTase اﻟﺤﺮ. . ﻟﻘﺪ وﺟﺪ أن اﻻﻣﺘﺼﺎص ﻣﺘﺴﺎوي اﻟﺤﺮارة ﻟـ CGTase ﻋﻠﻰ اﻷطﺮ اﻟﻌﻀﻮﯾﺔ اﻟﻤﻌﺪﻧﯿﺔ اﻟﺘﻲ ﺗﻢ اﺧﺘﺒﺎرھﺎ ﺗﺘﻤﺜﻞ ﺑﺸﻜﻞ أﻓﻀﻞ ﺑﺎﺳﺘﺨﺪام ﻣﺘﺴﺎوي اﻟﺤﺮارة Langmuir، ﺣﯿﺚ وﺟﺪت أن ﺳﻌﺎت اﻻﻣﺘﺼﺎص اﻟﻘﺼﻮى ﺗﺼﻞ إﻟﻰ 21، 30.6، 37.5، 40 ﻣﻠﻐﻢ/ﺟﻢ ﻋﻠﻰ Ca-TMA، Cu-BTC، MIL-101 اﻟﺼﻐﯿﺮة اﻟﻤﺴﺎﻣﯿﺔ وGNP، ﻋﻠﻰ اﻟﺘﻮاﻟﻲ. ﺗﻢ ﺗﺤﺴﯿﻦ ﻗﺪرة اﻻﻣﺘﺼﺎص إﻟﻰ 49.5 ﻣﺠﻢ / ﺟﻢ ﻋﻠﻰ H-Cu-BTC. وﻛﺎﻧﺖ ھﺬه اﻟﻘﺪرة أﻋﻠﻰ ﺑﻜﺜﯿﺮ ﻣﻦ ﺗﻠﻚ اﻟﺘﻲ ﻟﻮﺣﻈﺖ ﺑﺎﺳﺘﺨﺪام اﻟﺰﯾﻮﻟﯿﺖ - Y اﻟﺘﻘﻠﯿﺪي، واﻟﺘﻲ ﻟﻢ ﺗﺘﺠﺎوز 6.1 ﻣﻠﻐﻢ/ﺟﻢ، ﺑﺎﻹﺿﺎﻓﺔ إﻟﻰ وﺳﺎﺋﻞ اﻟﺪﻋﻢ اﻷﺧﺮى اﻟﻤﺬﻛﻮرة ﻓﻲ اﻷدﺑﯿﺎت اﻟﺴﺎﺑﻘﺔ. ﻛﺸﻒ ﺗﻮﺻﯿﻒ اﻟﺪﻋﺎﻣﺎت اﻟﻔﺎرﻏﺔ ﺑﺎﺳﺘﺨﺪام ﻣﺰﯾﺞ ﻣﻦ ﺣﯿﻮد اﻷﺷﻌﺔ اﻟﺴﯿﻨﯿﺔ (XRD)، واﻟﻤﺠﮭﺮ اﻹﻟﻜﺘﺮوﻧﻲ اﻟﻤﺎﺳﺢ (SEM)، وﺗﺤﻠﯿﻞ اﻟﺠﺎذﺑﯿﺔ اﻟﺤﺮارﯾﺔ (TGA) أن ھﯿﺎﻛﻞ اﻷطﺮ اﻟﻤﻌﺪﻧﯿﺔ اﻟﻌﻀﻮﯾﺔ ظﻠﺖ ﺳﻠﯿﻤﺔ ﺑﻌﺪ ﺗﺠﻤﯿﺪ CGTase. وأﺷﺎر ﺗﻔﻜﻚ ﻧﻄﺎق اﻷﻣﯿﺪ I ﻓﻲ ﻣﻄﯿﺎﻓﯿﺔ اﻷﺷﻌﺔ اﻟﺤﻤﺮاء ﺑﺎﺳﺘﺨﺪام ﺗﺤﻮﯾﻞ ﻓﻮرﯾﯿﮫ إﻟﻰ أن ﺟﺰﯾﺌﺎت CGTase اﻟﺤﺮة ﺗﺤﺘﻮي ﻓﻲ اﻟﻐﺎﻟﺐ ﻋﻠﻰ ﺻﻔﺎﺋﺢ β (56% صفائح β، و 38.5% α-helix و 5.5% β-turns ) ﻣﻊ ﺗﻐﯿﺮ ﺗﺮﻛﯿﺒﮭﺎ ﻋﻠﻰ MIL-101 (84.1% صفائح β، و لم توجد أي α-helix، و 15.9% β-turns)، Ca-TMA (51.3% صفائح β ، و 37.5% α-helix و 11.2% β-turns )، و GNP (41.9% صفائح β، و 18.1% α-helix و 40% β-turns ). وﻗﺪ ﻟﻮﺣﻈﺖ أﯾﻀًﺎ ﺗﻐﯿﯿﺮات ﺷﻜﻠﯿﺔ أﻗﻞ ﺑﺎﺳﺘﺨﺪام دﻋﺎﺋﻢ Cu-BTC (44% صفائح β، و 37.5% α-helix و 11.2% β-turns )، و H-Cu-BTC (76.1% صفائح β، و 23.9% α-helix و لم توجد أي β-turns ). ﺗﻢ اﺧﺘﺒﺎر CGTase اﻟﻤﺜﺒﺖ ﻋﻠﻰ اﻷطﺮ اﻟﻌﻀﻮﯾﺔ اﻟﻤﻌﺪﻧﯿﺔ اﻟﻤﺨﺘﻠﻔﺔ ﻹﻧﺘﺎج CDs ﻣﻦ اﻟﻨﺸﺎ، وﺗﻢ اﻟﺘﺤﻘﯿﻖ ﻓﻲ اﻟﻨﺸﺎط اﻟﻨﺴﺒﻲ وﻗﺎﺑﻠﯿﺔ إﻋﺎدة اﻻﺳﺘﺨﺪام وﻗﯿﻮد ﻧﻘﻞ اﻟﻜﺘﻠﺔ. ﻛﺎن اﻟﻨﺸﺎط اﻟﻤﺤﺪد ﻟـ CGTase اﻟﺤﺮ اﻟﻤﺴﺘﺨﺪم ھﻮ 167 وﺣﺪة / ﻣﻠﻎ، واﻟﺬي اﻧﺨﻔﺾ ﻋﻨﺪ اﻟﺘﺜﺒﯿﺖ إﻟﻰ 28 و38 و65.2 و98.5 وﺣﺪة / ﻣﻠﻎ ﻣﻦ اﻟﺒﺮوﺗﯿﻦ ﻋﻠﻰ GNP وCa-TMA وCu-BTC وH-CU-BTC ﻋﻠﻰ اﻟﺘﻮاﻟﻲ. ﻛﺸﻔﺖ دراﺳﺎت ﻗﺎﺑﻠﯿﺔ إﻋﺎدة اﻻﺳﺘﺨﺪام أﻧﮫ ﺑﻨﺎءً ﻋﻠﻰ α-CD، أظﮭﺮ 101-MIL ﻧﺸﺎط إﻧﺰﯾﻢ ﻣﺘﺒﻘﻲ ﺑﻨﺴﺒﺔ 29%، واﻟﺬي ﺗﺤﺴﻦ ﻣﻊ اﻻرﺗﺒﺎط اﻟﺘﺴﺎھﻤﻲ ﻋﺒﺮ اﻟﺠﻠﻮﺗﺎراﻟﺪھﯿﺪ إﻟﻰ 40%، وأﻋﻄﻰ Ca-TMA ﻧﺸﺎط ﺑﻨﺴﺒﺔ 33% ، و أﻣﺎ GNP ﻓﻘﺪ أﻋﻄﻰ ﻧﺸﺎط ﺑﻨﺴﺒﺔ 74% ﺑﻌﺪ ﺛﻤﺎﻧﻲ دورات ﺗﻔﺎﻋﻞ. أدى اﻧﺤﺒﺎس CGTase داﺧﻞ H-Cu-BTC إﻟﻰ ﻧﺸﺎط CGTase اﻟﻤﺘﺒﻘﻲ ﺑﻨﺴﺒﺔ 87% ﺑﻌﺪ ﻋﺸﺮ دورات ﺗﻔﺎﻋﻞ، ﻣﻘﺎرﻧﺔ ﺑـ 70% ﻋﻠﻰ Cu-BTC اﻟﺼﻐﯿﺮة اﻟﻤﺴﺎﻣﯿﺔ، ووﺟﻮد اﻟﻤﺴﺎم اﻟﻜﺒﯿﺮة واﻟﻤﺴﺎم اﻟﻤﺘﻮﺳﻄﺔ أدت إﻟﻰ ﺗﻌﺰﯾﺰ ﻧﻘﻞ اﻟﻜﺘﻠﺔ ﻣﻦ 0.68 ﻓﻲ اﻟﺪﻗﯿﻘﺔ ﻋﺒﺮ MOFs ذات اﻟﻤﺴﺎﻣﯿﺔ اﻟﺼﻐﯿﺮة إﻟﻰ 0.89 ﻓﻲ اﻟﺪﻗﯿﻘﺔ ﻋﻠﻰ H-Cu-BTC ﻛﺒﯿﺮ اﻟﻤﺴﺎم، وﺑﺎﻟﺘﺎﻟﻲ ﺗﺤﺴﯿﻦ إﻧﺘﺎج CDs. ﺗﻘﺪم ھﺬه اﻷطﺮوﺣﺔ ﻣﻌﻠﻮﻣﺎت ﻋﻦ ﺗﺄﺛﯿﺮ ﺧﺼﺎﺋﺺ ال MOFs ﻋﻠﻰ أداء CGTase، واﻟﺘﻲ ﯾﻤﻜﻦ اﺳﺘﺨﺪاﻣﮭﺎ ﻓﻲ ﺗﻄﻮﯾﺮ ﻣﺤﻔﺰات ﺣﯿﻮﯾﺔ ﻗﻮﯾﺔ ﺗﻌﺘﻤﺪ ﻋﻠﻰ CGTase ﻟﻠﺘﻄﺒﯿﻘﺎت اﻟﺼﻨﺎﻋﯿﺔ.

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