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Strategy in manipulating transglycosylation activity of glycosyl hydrolase for oligosaccharide production

Abdul Manas NH, Md Illias R, Mahadi NM

Crit Rev Biotechnol, 2018 Mar;38(2):272-293.
PMID: 28683572 DOI: 10.1080/07388551.2017.1339664

BACKGROUND: The increasing market demand for oligosaccharides has intensified the need for efficient biocatalysts. Glycosyl hydrolases (GHs) are still gaining popularity as biocatalyst for oligosaccharides synthesis owing to its simple reaction and high selectivity.
PURPOSE: Over the years, research has advanced mainly directing to one goal; to reduce hydrolysis activity of GHs for increased transglycosylation activity in achieving high production of oligosaccharides.
DESIGN AND METHODS: This review concisely presents the strategies to increase transglycosylation activity of GHs for oligosaccharides synthesis, focusing on controlling the reaction equilibrium, and protein engineering. Various modifications of the subsites of GHs have been demonstrated to significantly modulate the hydrolysis and transglycosylation activity of the enzymes. The clear insight of the roles of each amino acid in these sites provides a platform for designing an enzyme that could synthesize a specific oligosaccharide product.
CONCLUSIONS: The key strategies presented here are important for future improvement of GHs as a biocatalyst for oligosaccharide synthesis.
Fulltext The characterisation of an alkali-stable maltogenic amylase from Bacillus lehensis G1 and improved malto-oligosaccharide production by hydrolysis suppression

Abdul Manas NH, Pachelles S, Mahadi NM, Illias RM

PLoS One, 2014;9(9):e106481.
PMID: 25221964 DOI: 10.1371/journal.pone.0106481

A maltogenic amylase (MAG1) from alkaliphilic Bacillus lehensis G1 was cloned, expressed in Escherichia coli, purified and characterised for its hydrolysis and transglycosylation properties. The enzyme exhibited high stability at pH values from 7.0 to 10.0. The hydrolysis of β-cyclodextrin (β-CD) produced malto-oligosaccharides of various lengths. In addition to hydrolysis, MAG1 also demonstrated transglycosylation activity for the synthesis of longer malto-oligosaccharides. The thermodynamic equilibrium of the multiple reactions was shifted towards synthesis when the reaction conditions were optimised and the water activity was suppressed, which resulted in a yield of 38% transglycosylation products consisting of malto-oligosaccharides of various lengths. Thin layer chromatography and high-performance liquid chromatography analyses revealed the presence of malto-oligosaccharides with a higher degree of polymerisation than maltoheptaose, which has never been reported for other maltogenic amylases. The addition of organic solvents into the reaction further suppressed the water activity. The increase in the transglycosylation-to-hydrolysis ratio from 1.29 to 2.15 and the increased specificity toward maltopentaose production demonstrated the enhanced synthetic property of the enzyme. The high transglycosylation activity of maltogenic amylase offers a great advantage for synthesising malto-oligosaccharides and rare carbohydrates.
Effects of electrospun nanofiber fabrications on immobilization of recombinant Escherichia coli for production of xylitol from glucose

Mohamad Sukri N, Abdul Manas NH, Jaafar NR, A Rahman R, Abdul Murad AM, Md Illias R

Enzyme Microb Technol, 2024 Jan;172:110350.
PMID: 37948908 DOI: 10.1016/j.enzmictec.2023.110350

A suitable nanofiber sheet was formulated and developed based on its efficacy in the immobilization of recombinant Escherichia coli (E. coli) to enhance xylitol production. The effects of different types of nanofibers and solvents on cell immobilization and xylitol production were studied. The most applicable nanofiber membrane was selected via preliminary screening of four types of nanofiber membrane, followed by the selection of six different solvents. Polyvinylidene fluoride (PVDF) nanofiber sheet synthesized using dimethylformamide (DMF) solvent was found to be the most suitable carrier for immobilization and xylitol production. The thin, beaded PVDF (DMF) nanofibers were more favourable for microbial adhesion, with the number of immobilized cells as high as 96 × 106 ± 3.0 cfu/ml. The attraction force between positively charged PVDF nanofibers and the negatively charged E. coli indicates that the electrostatic interaction plays a significant role in cell adsorption. The use of DMF has also produced PVDF nanofibers biocatalyst capable of synthesizing the highest xylitol concentration (2.168 g/l) and productivity (0.090 g/l/h) and 55-69% reduction in cell lysis compared with DMSO solvent and free cells. This finding suggests that recombinant E. coli immobilized on nanofibers shows great potential as a whole-cell biocatalyst for xylitol production.
Effects of oil substrate supplementation on production of prodigiosin by Serratia nematodiphila for dye-sensitized solar cell

Abdul Manas NH, Chong LY, Tesfamariam YM, Zulkharnain A, Mahmud H, Abang Mahmod DS, et al.

J Biotechnol, 2020 Jun 20;317:16-26.
PMID: 32348830 DOI: 10.1016/j.jbiotec.2020.04.011

Bacterial pigments are potential substitute of chemical photosensitizer for dye-sensitized solar cell (DSSC) due to its non-toxic property and cost-effective production from microbial fermentation. Serratia nematodiphila YO1 was isolated from waterfall in Malaysia and identified using 16S ribosomal RNA. Characterization of the red pigment produced by the bacteria has confirmed the pigment as prodigiosin. Prodigiosin was produced from the fermentation of the bacteria in the presence of different oil substrates. Palm oil exhibited the best performance of cell growth and equivalent prodigiosin yield compared to olive oil and peanut oil. Prodigiosin produced with palm oil supplementation was 93 mg/l compared to 7.8 mg/l produced without supplementation, which recorded 11.9 times improvement. Specific growth rate of the cells improved 1.4 times when palm oil was supplemented in the medium. The prodigiosin pigment produced showed comparable performance as a DSSC sensitizer by displaying an open circuit voltage of 336.1 mV and a maximum short circuit current of 0.098 mV/cm2. This study stands a novelty in proving that the production of prodigiosin is favorable in the presence of palm oil substrate with high saturated fat content, which has not been studied before. This is also among the first bacterial prodigiosin tested as photosensitizer for DSSC application.
Carbon nanomaterial properties help to enhance xylanase production from recombinant Kluyveromyces lactis through a cell immobilization method

Abdul Manaf SA, Mohamad Fuzi SFZ, Low KO, Hegde G, Abdul Manas NH, Md Illias R, et al.

Appl Microbiol Biotechnol, 2021 Nov;105(21-22):8531-8544.
PMID: 34611725 DOI: 10.1007/s00253-021-11616-0

Carbon nanomaterials, due to their catalytic activity and high surface area, have potential as cell immobilization supports to increase the production of xylanase. Recombinant Kluyveromyces lactis used for xylanase production was integrated into a polymeric gel network with carbon nanomaterials. Carbon nanomaterials were pretreated before cell immobilization with hydrochloric acid (HCl) treatment and glutaraldehyde (GA) crosslinking, which contributes to cell immobilization performance. Carbon nanotubes (CNTs) and graphene oxide (GO) were further screened using a Plackett-Burman experimental design. Cell loading and agar concentration were the most important factors in xylanase production with low cell leakage. Under optimized conditions, xylanase production was increased by more than 400% compared to free cells. Immobilized cell material containing such high cell densities may exhibit new and unexplored beneficial properties because the cells comprise a large fraction of the component. The use of carbon nanomaterials as a cell immobilization support along with the entrapment method successfully enhances the production of xylanase, providing a new route to improved bioprocessing, particularly for the production of enzymes. KEY POINTS: • Carbon nanomaterials (CNTs, GO) have potential as cell immobilization supports. • Entrapment in a polymeric gel network provides space for xylanase production. • Plackett-Burman design screen for the most important factor for cell immobilization.
Emergence of nanomaterials as potential immobilization supports for whole cell biocatalysts and cell toxicity effects

Abdul Manaf SA, Mohamad Fuzi SFZ, Abdul Manas NH, Md Illias R, Low KO, Hegde G, et al.

Biotechnol Appl Biochem, 2021 Dec;68(6):1128-1138.
PMID: 32969042 DOI: 10.1002/bab.2034

The traditional approach of fermentation by a free cell system has limitations of low productivity and product separation that need to be addressed for production enhancement and cost effectiveness. One of potential methods to solve the problems is cell immobilization. Microbial cell immobilization allows more efficient up-scaling by reducing the nonproductive growth phase, improving product yield and simplifying product separation. Furthermore, the emergence of nanomaterials such as carbon nanotubes, graphene, and metal-based nanomaterials with excellent functional properties provides novel supports for cell immobilization. Nanomaterials have catalytic properties that can provide specific binding site with targeted cells. However, the toxicity of nanomaterials towards cells has hampered its application as it affects the biological system of the cells, which cannot be neglected in any way. This gray area in immobilization is an important concern that needs to be addressed and understood by researchers. This review paper discusses an overview of nanomaterials used for cell immobilization with special focus on its toxicological challenges and how by understanding physicochemical properties of nanomaterials could influence the toxicity and biocompatibility of the cells.