A Comparison of the Transglycosylation Capacity between the Guar GH27 Aga27A and Bacteroides GH36 BoGal36A α-Galactosidases
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals
2.2. Expression and Purification of BoGal 36A and Confirmation of Identity for Aga27A
2.3. Enzyme Activity Determination
2.4. Screening for Transglycosylation with pNP-Gal and MALDI-TOF MS Analysis
2.5. Screening for Transglycosylation with Raffinose or LBG and MALDI-TOF MS Analysis
2.6. Enzyme Stability in the Presence of Acceptors
2.7. Screening of Transglycosylation Products with Raffinose and LBG using HPLC
2.8. Evaluation of Transglycosylation Capacity with Raffinose or LBG and Methanol using HPLC
3. Results and Discussion
3.1. Enzyme Preparation
3.2. Initial Screening for Transglycosylation Ability Using pNP-Gal as Donor
3.3. Screening for the Ability to Utilzse Raffinose as the Donor Substrate
3.4. Screening for the Ability to Utilize Locust Bean Gum as the Donor Substrate
3.5. Analysis of Transglycosylation Products of BoGal36A and Aga27A Using HPLC
3.6. Stability in the Presence of Acceptor Molecules
3.7. Evaluation of Transglycosylation Capacity with Natural Substrates
3.7.1. Comparison of Transglycosylation Properties with Raffinose and Locust Bean Gum
3.7.2. Determination of Initial rS/rH Ratios and Assessment of Secondary Hydrolysis in Raffinose and Methanol Reactions
3.8. Evaluation of Transglycosylation Capacity Aga27A and BoGal36A
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Acceptor | BoGal36A | Aga27A | Product |
---|---|---|---|
pNP-Gal | N.D. | + | pNP-Gal-Gal |
10% Methanol | + | + | Methyl-Gal |
5% Propanol | + | + | Propyl-Gal |
10% Glycerol | + | + | Glyceryl-Gal |
5% Allyl-OH | + | + | Allyl-Gal |
2.5% Propargyl-OH | + | + | Propargyl-Gal |
Acceptor | BoGal36A | Aga27A | Product |
---|---|---|---|
Raffinose | + | + | Raffinose-Gal |
10% Methanol | + | + | Methyl-Gal |
5% Propanol | + | + | Propyl-Gal |
10% Glycerol | + | + | Glycerol-Gal |
5% Allyl-OH | N.D. | + | Allyl-Gal |
2.5% Propargyl-OH | N.D. | + | Propargyl-Gal |
Acceptor | BoGal36A | Aga27A | Product |
---|---|---|---|
10% Methanol | + | + | Methyl-Gal |
5% Propanol | N.D. | + | Propyl-Gal |
10% Glycerol | N.D. | + | Glycerol-Gal |
5% Allyl-OH | N.D. | + | Allyl-Gal |
2.5% Propargyl-OH | N.D. | N.D. | Propargyl-Gal |
Donor | Enzyme | [Me-Gal] (mM) | [Gal] (mM) | Product Yield (%) ([Me-Gal]/([Me-Gal]+[Gal])) | rS/rH ([Me-Gal]/[Gal]) |
---|---|---|---|---|---|
0.4 w/v% LBG | BoGal36A (24 h) | 0.7 (±0.1) | 2.1 (±0.4) | 27 | 0.33 (±0.06) |
Aga27A (24 h) | 1.0 (±0.1) | 2.4 (±0.1) | 30 | 0.42 (±0.05) | |
400 mM raffinose | BoGal36A (24 h) | 41 (±7) | 70 (±11) | 37 | 0.58 (±0.01) |
Aga27A (24 h) | 35 (±11) | 55 (±13) | 39 | 0.63 (±0.05) | |
40 mM raffinose | BoGal36A (30 min) | 1.3 (±0.1) | 1.4 (±0.2) | 48 | 0.99 (±0.06) |
BoGal36A (2h) | 3.1 (±0.1) | 4.2 (±0.3) | 42 | 0.74 (±0.07) | |
Aga27A (30 min) | 2.3 (±0.3) | 3.3 (±0.4) | 41 | 0.71 (±0.03) | |
Aga27A (3 h) | 4.0 (±0.6) | 6.4 (±1.0) | 38 | 0.63 (±0.04) |
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Wiemann, M.; Axell, E.; Stålbrand, H. A Comparison of the Transglycosylation Capacity between the Guar GH27 Aga27A and Bacteroides GH36 BoGal36A α-Galactosidases. Appl. Sci. 2022, 12, 5123. https://doi.org/10.3390/app12105123
Wiemann M, Axell E, Stålbrand H. A Comparison of the Transglycosylation Capacity between the Guar GH27 Aga27A and Bacteroides GH36 BoGal36A α-Galactosidases. Applied Sciences. 2022; 12(10):5123. https://doi.org/10.3390/app12105123
Chicago/Turabian StyleWiemann, Mathias, Emil Axell, and Henrik Stålbrand. 2022. "A Comparison of the Transglycosylation Capacity between the Guar GH27 Aga27A and Bacteroides GH36 BoGal36A α-Galactosidases" Applied Sciences 12, no. 10: 5123. https://doi.org/10.3390/app12105123
APA StyleWiemann, M., Axell, E., & Stålbrand, H. (2022). A Comparison of the Transglycosylation Capacity between the Guar GH27 Aga27A and Bacteroides GH36 BoGal36A α-Galactosidases. Applied Sciences, 12(10), 5123. https://doi.org/10.3390/app12105123