Publications by authors named "Rosli Md Illias"

67 Publications

Carbon nanomaterial properties help to enhance xylanase production from recombinant Kluyveromyces lactis through a cell immobilization method.

Appl Microbiol Biotechnol 2021 Oct 6. Epub 2021 Oct 6.

Faculty of Electrical and Electronic Engineering, Universiti Tun Hussein Onn Malaysia, 86400, Batu Pahat, Johor, Malaysia.

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.
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http://dx.doi.org/10.1007/s00253-021-11616-0DOI Listing
October 2021

Efficient substrate accessibility of cross-linked levanase aggregates using dialdehyde starch as a macromolecular cross-linker.

Carbohydr Polym 2021 Sep 7;267:118159. Epub 2021 May 7.

Department of Bioprocess Engineering, School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, 81300, Skudai, Johor Bahru, Johor Darul Takzim, Malaysia; Health and Wellness Research Alliance, Universiti Teknologi Malaysia, 81300, Skudai, Johor Bahru, Johor Darul Takzim, Malaysia; Institute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia. Electronic address:

Cross-linked enzyme aggregates (CLEAs) are influenced by mass diffusion limitations such as the degree of molecular cross-linking attained, which affects substrate accessibility. Thus, this study seeks to improve substrate accessibility using macromolecular cross-linkers in cross-linked levanase aggregates (CLLAs) formation for levan-type fructooligosaccharides (L-FOS) production. Dialdehyde starch-tapioca (DAST) was successfully developed and used to cross-link levanase to form CLLAs-D and with bovine serum albumin (BSA) to form CLLAs-DB which showed activity recoveries of 65.6% and 81.6%, respectively. After cross-linking, the pH (6-10) and thermal stability (30-40 °C) increased, and organic solvent tolerance resulted in the activation of CLLAs. Likewise, CLLAs-DB had higher substrate affinity and accessibility and a higher effectiveness factors than CLLAs-D. The total L-FOS yield of CLLAs-DB (78.9% (w/v)) was higher than that of CLLAs-D (62.4% (w/v)). Therefore, as a cross-linker, DAST may have application prospects as a promising and green biocatalyst for product formation.
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http://dx.doi.org/10.1016/j.carbpol.2021.118159DOI Listing
September 2021

Structural and functional characterisation of a cold-active yet heat-tolerant dehydroquinase from Glaciozyma antarctica PI12.

J Biotechnol 2021 Mar 1;329:118-127. Epub 2021 Feb 1.

Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor Darul Ehsan, Malaysia. Electronic address:

Dehydroquinase or 3-dehydroquinate dehydratase (DHQD) reversibly cleaves 3-dehydroquinate to form 3-dehydroshikimate. Here, we describe the functional and structural features of a cold active type II 3-dehydroquinate dehydratase from the psychrophilic yeast, Glaciozyma antarctica PI12 (GaDHQD). Functional studies showed that the enzyme was active at low temperatures (10-30 °C), but displayed maximal activity at 40 °C. Yet the enzyme was stable over a wide range of temperatures (10-70 °C) and between pH 6.0-10.0 with an optimum pH of 8.0. Interestingly, the enzyme was highly thermo-tolerant, denaturing only at approximately 84 °C. Three-dimensional structure analyses showed that the G. antarctica dehydroquinase (GaDHQD) possesses psychrophilic features in comparison with its mesophilic and thermophilic counterparts such as higher numbers of non-polar residues on the surface, lower numbers of arginine and higher numbers of glycine-residues with lower numbers of hydrophobic interactions. On the other hand, GaDHQD shares some traits (i.e. total number of hydrogen bonds, number of proline residues and overall folding) with its mesophilic and thermophilic counterparts. Combined, these features contribute synergistically towards the enzyme's ability to function at both low and high temperatures.
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http://dx.doi.org/10.1016/j.jbiotec.2021.01.019DOI Listing
March 2021

Optimization strategy for laccase immobilization on polyethylene terephthalate grafted with maleic anhydride electrospun nanofiber mat.

Int J Biol Macromol 2021 Jan 2;166:876-883. Epub 2020 Nov 2.

School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.

Enzyme immobilization has been known to be one of the methods to improve the stability and reusability of enzyme. In this study, a strategy to optimize laccase immobilization on polyethylene terephthalate grafted with maleic anhydride electrospun nanofiber mat (PET-g-MAH ENM) was developed. The development involves the screening and optimization processes of the crucial factors that influence the immobilization yield such as enzyme concentration, pH values, covalent bonding (CV) time, CV temperature, crosslinking (CL) time, CL temperature and glutaraldehyde concentration using two-level factorial design and Box-Behnken design (BBD), respectively. It was found that laccase concentration, pH values and glutaraldehyde concentration play important role in enhancing the immobilization yield of laccase on PET-g-MAH ENM in the screening process. Subsequently, the optimization result showed at 0.28 mg/ml laccase concentration, pH 3 and 0.45% (v/v) glutaraldehyde concentrations gave the highest immobilization yield at 87.64% which was 81.2% increment from the immobilization yield before optimization. Under the optimum condition, the immobilized laccase was able to oxidize 2, 2-azino-bis 3-ethylbenzothiazoline-6- sulfonic acid (ABTS) in a broad range of pH (pH 3-6) and temperature (20- 70 °C). Meanwhile, the kinetic parameters for K and V were 1.331 mM and 0.041 mM/min, respectively. It was concluded that the optimization of immobilized laccase on PET-g-MAH ENM enhance the performance of this biocatalyst.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.10.244DOI Listing
January 2021

Emergence of nanomaterials as potential immobilization supports for whole cell biocatalysts and cell toxicity effects.

Biotechnol Appl Biochem 2020 Sep 24. Epub 2020 Sep 24.

Freshwater Aquaculture Center-College of Fisheries, Central Luzon State University, Nueva Ecija, Philippines.

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.
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http://dx.doi.org/10.1002/bab.2034DOI Listing
September 2020

Functional characterisation and product specificity of Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1.

Enzyme Microb Technol 2020 Oct 20;140:109625. Epub 2020 Jun 20.

Institute of Bioproduct Developments, Universiti Teknologi Malaysia, 81310, Skudai, Johor, Malaysia. Electronic address:

Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1 (Blg32) composed of 284 amino acids with a predicted molecular mass of 31.6 kDa is expressed in Escherichia coli and purified to homogeneity. Herein, Blg32 characteristics, substrates and product specificity as well as structural traits that might be involved in the production of sugar molecules are analysed. This enzyme functions optimally at the temperature of 70 °C, pH value of 8.0 with its catalytic activity strongly enhanced by Mn. Remarkably, the purified enzyme is highly stable in high temperature and alkaline conditions. It exhibits the highest activity on laminarin (376.73 U/mg) followed by curdlan and yeast β-glucan. Blg32 activity increased by 62% towards soluble substrate (laminarin) compared to insoluble substrate (curdlan). Hydrolytic products of laminarin were oligosaccharides with degree of polymerisation (DP) of 1 to 5 with the main product being laminaritriose (DP3). This suggests that the active site of Blg32 could recognise up to five glucose units. High concentration of Blg32 mainly produces glucose whilst low concentration of Blg32 yields oligosaccharides with different DP (predominantly DP3). A theoretical structural model of Blg32 was constructed and structural analysis revealed that Trp156 is involved in multiple hydrophobic stacking interactions. The amino acid was predicted to participate in substrate recognition and binding. It was also exhibited that catalytic groove of Blg32 has a narrow angle, thus limiting the substrate binding reaction. All these properties and knowledge of the subsites are suggested to be related to the possible mode of action of how Blg32 produces glucooligosaccharides.
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http://dx.doi.org/10.1016/j.enzmictec.2020.109625DOI Listing
October 2020

Novel cross-linked enzyme aggregates of levanase from Bacillus lehensis G1 for short-chain fructooligosaccharides synthesis: Developmental, physicochemical, kinetic and thermodynamic properties.

Int J Biol Macromol 2020 Sep 4;159:577-589. Epub 2020 May 4.

School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, 81300, Skudai, Johor Bahru, Johor Darul Takzim, Malaysia; Health and Wellness Research Alliance, Universiti Teknologi Malaysia, 81310, UTM Skudai, Johor Bahru, Malaysia; Institute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia. Electronic address:

Short-chain fructooligosaccharides (scFOSs) can be produced from the levan hydrolysis using levanase. Levanase from Bacillus lehensis G1 (rlevblg1) is an enzyme that specifically converts levan to scFOSs. However, the use of free levanase presents a lack of stability and reusability, thus hindering the synthesis of scFOSs for continuous reactions. Here, CLEAs for rlevblg1 were prepared and characterized. Cross-linked levanase aggregates using glutaraldehyde (CLLAs-ga) and bovine albumin serum (CLLAs-ga-bsa) showed the best activity recovery of 92.8% and 121.2%, respectively. The optimum temperature of CLLAs-ga and CLLAs-ga-bsa was increased to 35 °C and 40 °C, respectively, from its free rlevblg1 (30 °C). At high temperature (50 °C), the half-life of CLLAs-ga-bsa was higher than that of free rlevblg1 and CLLAs-ga. Both CLLAs exhibited higher stability at pH 9 and pH 10. Hyperactivation of CLLAs-ga-bsa was achieved with an effectiveness factor of more than 1 and with improved catalytic efficiency. After 3 h reaction, CLLAs-ga-bsa produced the highest total scFOSs yield of 35.4% and total sugar of 60.4% per gram levan. Finally, the reusability of CLLAs for 8 cycles with more than 50% activity retained makes them as a potential synthetic catalyst to be explored for scFOSs synthesis.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.04.262DOI Listing
September 2020

Immobilization of recombinant Escherichia coli on multi-walled carbon nanotubes for xylitol production.

Enzyme Microb Technol 2020 Apr 23;135:109495. Epub 2019 Dec 23.

School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia; Institute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia. Electronic address:

E. coli has been engineered to produce xylitol, but the production faces bottlenecks in terms of production yield and cell viability. In this study, recombinant E. coli (rE. coli) was immobilized on untreated and treated multiwalled carbon nanotubes (MWCNTs) for xylitol production. The immobilized rE. coli on untreated MWCNTs gave the highest xylitol production (5.47 g L) and a productivity of 0.22 g L h. The doubling time for the immobilized cells increased up to 20.40 h and was higher than that of free cells (3.67 h). Cell lysis of the immobilized cells was reduced by up to 73 %, and plasmid stability improved by up to 17 % compared to those of free cells. Xylitol production using the optimum parameters (pH 7.4, 0.005 mM and 29 °C) achieved a xylitol production and productivity of 6.33 g L and 0.26 g L h, respectively. A seven-cycle repeated batch fermentation was carried out for up to 168 h, which showed maximum xylitol production of 7.36 g L during the third cycle. Hence, this new adsorption immobilization system using MWCNTs is an alternative to improve the production of xylitol.
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http://dx.doi.org/10.1016/j.enzmictec.2019.109495DOI Listing
April 2020

Entrapment of porous cross-linked enzyme aggregates of maltogenic amylase from Bacillus lehensis G1 into calcium alginate for maltooligosaccharides synthesis.

Int J Biol Macromol 2020 May 5;150:80-89. Epub 2020 Feb 5.

School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia; Institute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia. Electronic address:

Maltooligosaccharides (MOSs) are emerging oligosaccharides in food-based applications and can be synthesized through the enzymatic synthesis of maltogenic amylase from Bacillus lehensis G1 (Mag1). However, the lack of enzyme stability makes this approach unrealistic for industrial applications. The formation of cross-linked enzyme aggregates (CLEAs) is a promising tool for improving enzyme stability, and the substrate accessibility problem of CLEA formation was overcome by the addition of porous agents to generate porous CLEAs (p-CLEAs). However, p-CLEAs exhibited high enzyme leaching and low solvent tolerance. To address these problems, p-CLEAs of Mag1 (Mag1-p-CLEAs) were entrapped in calcium alginate beads (CA). Mag1-p-CLEAs-CA prepared with 2.5% (w/v) sodium alginate and 0.6% (w/v) calcium chloride yielded 53.16% (17.0 U/mg) activity and showed a lower deactivation rate and longer half-life than those of entrapped free Mag1 (Mag1-CA) and entrapped non-porous Mag1-CLEAs (Mag1-CLEAs-CA). Moreover, Mag1-p-CLEAs-CA exhibited low enzyme leaching and high tolerance in various solvents compared to Mag1-p-CLEAs. A kinetic study revealed that Mag1-p-CLEAs-CA exhibited relatively high affinity towards beta-cyclodextrin (β-CD) (K = 0.62 mM). MOSs (300 mg/g) were synthesized by Mag1-p-CLEAs-CA at 50 °C. Finally, the reusability of Mag1-p-CLEAs-CA makes them as a potential biocatalyst for the continuous synthesis of MOSs.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.02.032DOI Listing
May 2020

Functional and structural analyses of an expansin-like protein from the antarctic yeast Glaciozyma antarctica PI12 reveal strategies of nutrient scavenging in the sea ice environment.

Int J Biol Macromol 2020 Feb 13;144:231-241. Epub 2019 Dec 13.

School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, UKM, 43600 Bangi, Selangor, Malaysia. Electronic address:

Genome data mining of the Antarctic yeast, Glaciozyma antarctica PI12 revealed an expansin-like protein encoding sequence (GaEXLX1). The GaEXLX1 protein is 24.8 kDa with a high alkaline pI of 9.81. Homology modeling of GaEXLX1 showed complete D1 and D2 domains of a conventional expansin. The protein exhibited 36% sequence similarity to Clavibacter michiganensis EXLX1 (PDB: 4JCW). Subsequently, a recombinant GaEXLX1 protein was produced using Escherichia coli expression system. Incubation with Avicel, filter paper and cotton fiber showed that the protein can disrupt the surface of crystalline and pure cellulose, suggesting a cell wall modification activity usually exhibited by expansin-like proteins. Binding assays displayed that GaEXLX1 can bind to polymeric substrates, including those postulated to be present in the sea ice ecosystem such as crab chitin and moss lichenan. GaEXLX1 may assist in the recognition and loosening of these substrates in the sea ice prior to hydrolysis by other extracellular enzymes. Similar loosening mechanism to classical expansin-like protein has been postulated for this psychrophilic protein based on several conserved residues of GaEXLX1 involved in binding interaction identified by docking analyses.
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http://dx.doi.org/10.1016/j.ijbiomac.2019.12.099DOI Listing
February 2020

A porous-cross linked enzyme aggregates of maltogenic amylase from Bacillus lehensis G1: Robust biocatalyst with improved stability and substrate diffusion.

Int J Biol Macromol 2020 Apr 20;148:1222-1231. Epub 2019 Nov 20.

School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, Skudai, Johor 81310, Malaysia; Institute of Bioproduct Development, Universiti Teknologi Malaysia, Skudai, Johor 81310, Malaysia. Electronic address:

Enzymatic synthesis of maltooligosaccharides is hampered due to lack of stability of soluble enzyme. This limitation can be tackled by cross linked enzyme aggregates (CLEAs) immobilization approach. However, substrate diffusion is a major bottleneck in cross linking technology. Herein, CLEAs of maltogenic amylase from Bacillus lehensis G1 (Mag1) was developed with addition of porous agent (Mag1-p-CLEAs). Comparison of thermal, pH and kinetic analysis with CLEAs without porous agent (Mag1-CLEAs) and free Mag1 was performed. Mag1-p-CLEAs with porous structure prepared at 0.8% (w/v) of citrus pectin (porous agent), 0.25% (w/v) of chitosan (cross linker) and cross linked for 1.5 h yielded 91.20% activity. 80% of activity is retained after 30 min of incubation at 40 °C and showed longer half-life than free Mag1 and Mag1-CLEAs. Mag1-p-CLEAs also showed pH stability at acidic and alkaline pH. The 1.68-fold increase in V value in comparison to Mag1-CLEAs showed that the presence of pores of Mag1-p-CLEAs enhanced the beta-cyclodextrin accessibility. The increase in high catalytic efficiency (K/K) value, 1.90-fold and 1.05-fold showed that it also has better catalytic efficiency than free Mag1 and Mag1-CLEAs, respectively. Mag1-p-CLEAs not only improved substrate diffusibility of CLEAs, but also leads to higher thermal and pH stability of Mag1.
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http://dx.doi.org/10.1016/j.ijbiomac.2019.10.101DOI Listing
April 2020

Protein engineering of GH11 xylanase from Aspergillus fumigatus RT-1 for catalytic efficiency improvement on kenaf biomass hydrolysis.

Enzyme Microb Technol 2019 Dec 17;131:109383. Epub 2019 Jul 17.

School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia; Institute of Bioproduct Development, Universiti Teknologi Makaysia, 81310 Skudai, Johor, Malaysia. Electronic address:

Enzyme hydrolysis faces a bottleneck due to the recalcitrance of the lignocellulose biomass. The protein engineering of GH11 xylanase from Aspergillus fumigatus RT-1 was performed near the active site and at the N-terminal region to improve its catalytic efficiency towards pretreated kenaf (Hibiscus cannabinus) hydrolysis. Five mutants were constructed by combined approaches of error-prone PCR, site-saturation and site-directed mutagenesis. The double mutant c168 t/Q192H showed the most effective hydrolysis reaction with a 13.9-fold increase in catalytic efficiency, followed by mutants Y7L and c168 t/Q192 H/Y7L with a 1.6-fold increase, respectively. The enhanced catalytic efficiency evoked an increase in sugar yield of up to 28% from pretreated kenaf. In addition, mutant c168 t/Q192 H/Y7L improved the thermostability at higher temperature and acid stability. This finding shows that mutations at distances less than 15 Å from the active site and at putative secondary binding sites affect xylanase catalytic efficiency towards insoluble substrates hydrolysis.
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http://dx.doi.org/10.1016/j.enzmictec.2019.109383DOI Listing
December 2019

Enhanced secretion of cyclodextrin glucanotransferase (CGTase) by Lactococcus lactis using heterologous signal peptides and optimization of cultivation conditions.

J Biotechnol 2019 Apr 13;296:22-31. Epub 2019 Mar 13.

Department of Bioprocess and Polymer Engineering, School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, 81310, Skudai, Johor, Malaysia. Electronic address:

In previous studies of Lactococcus lactis, the levels of proteins secreted using heterologous signal peptides were observed to be lower than those obtained using the signal peptide from Usp45, the major secreted lactococcal protein. In this study, G1 (the native signal peptide of CGTase) and the signal peptide M5 (mutant of the G1 signal peptide) were introduced into L. lactis to investigate the effect of signal peptides on lactococcal protein secretion to improve secretion efficiency. The effectiveness of these signal peptides were compared to the Usp45 signal peptide. The highest secretion levels were obtained using the G1 signal peptide. Sequence analysis of signal peptide amino acids revealed that a basic N-terminal signal peptide is not absolutely required for efficient protein export in L. lactis. Moreover, the introduction of a helix-breaking residue in the H-region of the M5 signal peptide caused a reduction in the signal peptide hydrophobicity and decreased protein secretion. In addition, the optimization of cultivation conditions for recombinant G1-CGTase production via response surface methodology (RSM) showed that CGTase activity increased approximately 2.92-fold from 5.01 to 16.89 U/ml compared to the unoptimized conditions.
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http://dx.doi.org/10.1016/j.jbiotec.2019.02.013DOI Listing
April 2019

Preparation of kenaf stem hemicellulosic hydrolysate and its fermentability in microbial production of xylitol by Escherichia coli BL21.

Sci Rep 2019 03 11;9(1):4080. Epub 2019 Mar 11.

Chemical Engineering Programme, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor Darul Ehsan, Malaysia.

Kenaf (Hibiscus cannabinus L.), a potential fibre crop with a desirably high growth rate, could serve as a sustainable feedstock in the production of xylitol. In this work, the extraction of soluble products of kenaf through dilute nitric-acid hydrolysis was elucidated with respect to three parameters, namely temperature, residence time, and acid concentration. The study will assist in evaluating the performance in terms of xylose recovery. The result point out that the maximum xylose yield of 30.7 g per 100 g of dry kenaf was attained from 2% (v/v) HNO at 130 °C for 60 min. The detoxified hydrolysate was incorporated as the primary carbon source for subsequent fermentation by recombinant Escherichia coli and the performance of strain on five different semi-synthetic media on xylitol production were evaluated herein. Among these media, batch cultivation in a basal salt medium (BSM) afforded the highest xylitol yield of 0.35 g/g based on xylose consumption, which corresponded to 92.8% substrate utilization after 38 h. Subsequently, fermentation by E. coli in the xylose-based kenaf hydrolysate supplemented with BSM resulting in 6.8 g/L xylitol which corresponding to xylitol yield of 0.38 g/g. These findings suggested that the use of kenaf as the fermentation feedstock could be advantageous for the development of sustainable xylitol production.
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http://dx.doi.org/10.1038/s41598-019-40807-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411968PMC
March 2019

Comparative structural analysis of fruit and stem bromelain from Ananas comosus.

Food Chem 2018 Nov 30;266:183-191. Epub 2018 May 30.

Department of Bioprocess and Polymer Engineering, Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.

Cysteine proteases in pineapple (Ananas comosus) plants are phytotherapeutical agents that demonstrate anti-edematous, anti-inflammatory, anti-thrombotic and fibrinolytic activities. Bromelain has been identified as an active component and as a major protease of A. comosus. Bromelain has gained wide acceptance and compliance as a phytotherapeutical drug. The proteolytic fraction of pineapple stem is termed stem bromelain, while the one presents in the fruit is known as fruit bromelain. The amino acid sequence and domain analysis of the fruit and stem bromelains demonstrated several differences and similarities of these cysteine protease family members. In addition, analysis of the modelled fruit (BAA21848) and stem (CAA08861) bromelains revealed the presence of unique properties of the predicted structures. Sequence analysis and structural prediction of stem and fruit bromelains of A. comosus along with the comparison of both structures provides a new insight on their distinct properties for industrial application.
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http://dx.doi.org/10.1016/j.foodchem.2018.05.125DOI Listing
November 2018

Secretome analysis of alkaliphilic bacterium Bacillus lehensis G1 in response to pH changes.

Microbiol Res 2018 Oct 19;215:46-54. Epub 2018 Jun 19.

Department of Bioprocess and Polymer Engineering, Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia. Electronic address:

Bacillus lehensis G1 is an alkaliphilic bacterium that is capable of surviving in environments up to pH 11. Secretome related to bacterial acclimation in alkaline environment has been less studied compared to cytoplasmic and membrane proteome. The aim of this study was to gain better understanding of bacterial acclimation to alkaline media through analyzing extracellular proteins of B. lehensis. The pH range for B. lehensis growth was conducted, and two-dimensional electrophoresis and MALDI-TOF/TOF MS analysis were conducted to characterize changes in protein profiling in B. lehensis cultured at pH 8 and pH 11 when compared with those cultured at pH 10 (optimal growth pH). B. lehensis could grow well at pH ranging from 8 to 11 in which the bacteria showed to posses thinner flagella at pH 11. Proteomic analyses demonstrated that five proteins were up-regulated and 13 proteins were down-regulated at pH 8, whereas at pH 11, 14 proteins were up-regulated and 8 were down-regulated. Majority of the differentially expressed proteins were involved in the cell wall, main glycolytic pathways, the metabolism of amino acids and related molecules and some proteins of unknown function. A total of 40 differentially expressed protein spots corresponding to 33 proteins were identified; including GlcNAc-binding protein A, chitinase, endopeptidase lytE, flagellar hook-associated proteins and enolase. These proteins may play important roles in acclimation to alkaline media via reallocation of cell wall structure and changes to cell surface glycolytic enzymes, amino acid metabolism, flagellar hook-associated proteins and chaperones to sustain life under pH-stressed conditions.
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http://dx.doi.org/10.1016/j.micres.2018.06.006DOI Listing
October 2018

Unravelling the adaptation strategies employed by Glaciozyma antarctica PI12 on Antarctic sea ice.

Mar Environ Res 2018 Jun 21;137:169-176. Epub 2018 Mar 21.

School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor, Malaysia.

Glaciozyma antarctica PI12, is a psychrophilic yeast isolated from Antarctic sea. In this work, Expressed Sequence Tags (EST) from cells exposed to three different temperatures; 15 °C, 0 °C and -12 °C were generated to identify genes associated with cold adaptation. A total of 5376 clones from each library were randomly picked and sequenced. Comparative analyses from the resulting ESTs in each condition identified several groups of genes required for cold adaptation. Additionally, 319 unique transcripts that encoded uncharacterised functions were identified in the -12 °C library and are currently unique to G. antarctica. Gene expression analysis using RT-qPCR revealed two of the unknown genes to be up-regulated at -12 °C compared to 0 °C and 15 °C. These findings further contribute to the collective knowledge into G. antarctica cold adaptation and as a resource for understanding the ecological and physiological tolerance of psychrophilic microbes in general.
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http://dx.doi.org/10.1016/j.marenvres.2018.03.007DOI Listing
June 2018

Biochemical and structural characterization of a novel cold-active esterase-like protein from the psychrophilic yeast Glaciozyma antarctica.

Extremophiles 2018 Jul 20;22(4):607-616. Epub 2018 Mar 20.

School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia.

Dienelactone hydrolase, an α/β hydrolase enzyme, catalyzes the hydrolysis of dienelactone to maleylacetate, an intermediate for the Krebs cycle. Genome sequencing of the psychrophilic yeast, Glaciozyma antarctica predicted a putative open reading frame (ORF) for dienelactone hydrolase (GaDlh) with 52% sequence similarity to that from Coniophora puteana. Phylogenetic tree analysis showed that GaDlh is closely related to other reported dienelactone hydrolases, and distantly related to other α/β hydrolases. Structural prediction using MODELLER 9.14 showed that GaDlh has the same α/β hydrolase fold as other dienelactone hydrolases and esterase/lipase enzymes, with a catalytic triad consisting of Cys-His-Asp and a G-x-C-x-G-G motif. Based on the predicted structure, GaDlh exhibits several characteristics of cold-adapted proteins such as glycine clustering in the binding pocket, reduced protein core hydrophobicity, and the absence of proline residues in loops. The putative ORF was amplified, cloned, and overexpressed in an Escherichia coli expression system. The recombinant protein was overexpressed as soluble proteins and was purified via Ni-NTA affinity chromatography. Biochemical characterization of GaDlh revealed that it has an optimal temperature at 10 °C and that it retained almost 90% of its residual activity when incubated for 90 min at 10 °C. The optimal pH was at pH 8.0 and it was stable between pH 5-9 when incubated for 60 min (more than 50% residual activity). Its K value was 256 μM and its catalytic efficiency was 81.7 s. To our knowledge, this is the first report describing a novel cold-active dienelactone hydrolase-like protein.
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http://dx.doi.org/10.1007/s00792-018-1021-zDOI Listing
July 2018

The Glaciozyma antarctica genome reveals an array of systems that provide sustained responses towards temperature variations in a persistently cold habitat.

PLoS One 2018 31;13(1):e0189947. Epub 2018 Jan 31.

Malaysia Genome Institute, Jalan Bangi Lama, Kajang, Selangor, Malaysia.

Extremely low temperatures present various challenges to life that include ice formation and effects on metabolic capacity. Psyhcrophilic microorganisms typically have an array of mechanisms to enable survival in cold temperatures. In this study, we sequenced and analysed the genome of a psychrophilic yeast isolated in the Antarctic region, Glaciozyma antarctica. The genome annotation identified 7857 protein coding sequences. From the genome sequence analysis we were able to identify genes that encoded for proteins known to be associated with cold survival, in addition to annotating genes that are unique to G. antarctica. For genes that are known to be involved in cold adaptation such as anti-freeze proteins (AFPs), our gene expression analysis revealed that they were differentially transcribed over time and in response to different temperatures. This indicated the presence of an array of adaptation systems that can respond to a changing but persistent cold environment. We were also able to validate the activity of all the AFPs annotated where the recombinant AFPs demonstrated anti-freeze capacity. This work is an important foundation for further collective exploration into psychrophilic microbiology where among other potential, the genes unique to this species may represent a pool of novel mechanisms for cold survival.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0189947PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5791967PMC
February 2018

Site-saturation mutagenesis of mutant L-asparaginase II signal peptide hydrophobic region for improved excretion of cyclodextrin glucanotransferase.

J Ind Microbiol Biotechnol 2017 Dec 18;44(12):1627-1641. Epub 2017 Sep 18.

Department of Bioprocess and Polymer Engineering, Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, 81310, Skudai, Johor, Malaysia.

The excretion of cyclodextrin glucanotransferase (CGTase) into the culture medium offers significant advantages over cytoplasmic expression. However, the limitation of Escherichia coli is its inability to excrete high amount of CGTase outside the cells. In this study, modification of the hydrophobic region of the N1R3 signal peptide using site-saturation mutagenesis improved the excretion of CGTase. Signal peptide mutants designated M9F, V10L and A15Y enhanced the excretion of CGTase three-fold and demonstrated two-fold higher secretion rate than the wild type. However, high secretion rate of these mutants was non-productive for recombinant protein production because it caused up to a seven-fold increase in cell death compared to the wild type. Our results indicated that the excretion of CGTase is highly dependent on hydrophobicity, secondary conformation and the type and position of amino acids at the region boundary and core segment of the h-region.
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http://dx.doi.org/10.1007/s10295-017-1980-6DOI Listing
December 2017

Data for proteome analysis of G1 in starch-containing medium.

Data Brief 2017 Oct 14;14:35-40. Epub 2017 Jul 14.

Department of Bioprocess and Polymer Engineering, Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.

G1 is a cyclodextrin glucanotransferase (CGTase) producer, which can degrade starch into cyclodextrin. Here, we present the proteomics data of cultured in starch-containing medium, which is related to the article "Proteome-based identification of signal peptides for improved secretion of recombinant cyclomaltodextrin glucanotransferase in " (Ling et. al, in press). This dataset was generated to better understand the secretion of proteins involved in starch utilization for bacterial sustained growth. A 2-DE proteomic technique was used and the proteins were tryptically digested followed by detection using MALDI-TOF/TOF. Proteins were classified into functional groups using the information available in SubtiList webserver (http://genolist.pasteur.fr/SubtiList/).
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http://dx.doi.org/10.1016/j.dib.2017.07.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524313PMC
October 2017

Strategy in manipulating transglycosylation activity of glycosyl hydrolase for oligosaccharide production.

Crit Rev Biotechnol 2018 Mar 6;38(2):272-293. Epub 2017 Jul 6.

d Comparative Genomics and Genetics Research Centre , Malaysia Genome Institute , Kajang , Malaysia.

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.
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http://dx.doi.org/10.1080/07388551.2017.1339664DOI Listing
March 2018

Biotechnological route for sustainable succinate production utilizing oil palm frond and kenaf as potential carbon sources.

Appl Microbiol Biotechnol 2017 Apr 9;101(8):3055-3075. Epub 2017 Mar 9.

Research Centre for Sustainable Process Technology (CESPRO), Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia.

Due to the world's dwindling energy supplies, greater thrust has been placed on the utilization of renewable resources for global succinate production. Exploration of such biotechnological route could be seen as an act of counterbalance to the continued fossil fuel dominance. Malaysia being a tropical country stands out among many other nations for its plenty of resources in the form of lignocellulosic biomass. To date, oil palm frond (OPF) contributes to the largest fraction of agricultural residues in Malaysia, while kenaf, a newly introduced fiber crop with relatively high growth rate, holds great potential for developing sustainable succinate production, apart from OPF. Utilization of non-food, inexhaustible, and low-cost derived biomass in the form of OPF and kenaf for bio-based succinate production remains largely untapped. Owing to the richness of carbohydrates in OPF and kenaf, bio-succinate commercialization using these sources appears as an attractive proposition for future sustainable developments. The aim of this paper was to review some research efforts in developing a biorefinery system based on OPF and kenaf as processing inputs. It presents the importance of the current progress in bio-succinate commercialization, in addition to describing the potential use of different succinate production hosts and various pretreatments-saccharifications under development for OPF and kenaf. Evaluations on the feasibility of OPF and kenaf as fermentation substrates are also discussed.
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http://dx.doi.org/10.1007/s00253-017-8210-zDOI Listing
April 2017

Crystal structure of fuculose aldolase from the Antarctic psychrophilic yeast Glaciozyma antarctica PI12.

Acta Crystallogr F Struct Biol Commun 2016 11 27;72(Pt 11):831-839. Epub 2016 Oct 27.

School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor Darul Ehsan 43600, Malaysia.

Fuculose-1-phosphate aldolase (FucA) catalyses the reversible cleavage of L-fuculose 1-phosphate to dihydroxyacetone phosphate (DHAP) and L-lactaldehyde. This enzyme from mesophiles and thermophiles has been extensively studied; however, there is no report on this enzyme from a psychrophile. In this study, the gene encoding FucA from Glaciozyma antarctica PI12 (GaFucA) was cloned and the enzyme was overexpressed in Escherichia coli, purified and crystallized. The tetrameric structure of GaFucA was determined to 1.34 Å resolution. The overall architecture of GaFucA and its catalytically essential histidine triad are highly conserved among other fuculose aldolases. Comparisons of structural features between GaFucA and its mesophilic and thermophilic homologues revealed that the enzyme has typical psychrophilic attributes, indicated by the presence of a high number of nonpolar residues at the surface and a lower number of arginine residues.
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http://dx.doi.org/10.1107/S2053230X16015612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5101584PMC
November 2016

Bromelain: from production to commercialisation.

J Sci Food Agric 2017 Mar 29;97(5):1386-1395. Epub 2016 Nov 29.

Department of Bioprocess Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81310, Skudai, Johor, Malaysia.

Bromelain is a mixture of proteolytic enzymes found in pineapple (Ananas comosus) plants. It can be found in several parts of the pineapple plant, including the stem, fruit, leaves and peel. High demand for bromelain has resulted in gradual increases in bromelain production. These increases have led to the need for a bromelain production strategy that yields more purified bromelain at a lower cost and with fewer production steps. Previously, bromelain was purified by conventional centrifugation, ultrafiltration and lyophilisation. Recently, the development of more modern purification techniques such as gel filtration, ion exchange chromatography, affinity chromatography, aqueous two-phase extraction and reverse micelle chromatography has resulted in increased industrial bromelain production worldwide. In addition, recombinant DNA technology has emerged as an alternative strategy for producing large amounts of ultrapure bromelain. An up-to-date compilation of data regarding the commercialisation of bromelain in the clinical, pharmaceutical and industrial fields is provided in this review. © 2016 Society of Chemical Industry.
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http://dx.doi.org/10.1002/jsfa.8122DOI Listing
March 2017

Expression and characterization of a cellobiohydrolase (CBH7B) from the thermophilic fungus Thielavia terrestris in Pichia pastoris.

Biotechnol Appl Biochem 2016 Sep 2;63(5):690-698. Epub 2015 Nov 2.

School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Selangor, Malaysia.

The gene encoding a cellobiohydrolase 7B (CBH7B) of the thermophilic fungus Thielavia terrestris was identified, subcloned, and expressed in Pichia pastoris. CBH7B encoded 455 amino acid residues with a molecular mass of 51.8 kDa. Domain analysis indicated that CBH7B contains a family 7 glycosyl hydrolase catalytic core but lacks a carbohydrate-binding module. Purified CBH7B exhibited optimum catalytic activity at pH 5.0 and 55 °C with 4-methylumbelliferryl-cellobioside as the substrate and retained 85% of its activity following 24 H incubation at 50 °C. Despite the lack of activity toward microcrystalline substrates, this enzyme worked synergistically with the commercial enzyme cocktail Cellic CTec2 to enhance saccharification by 39% when added to a reaction mixture containing 0.25% alkaline pretreated oil palm empty fruit bunch (OPEFB). Attenuated total reflectance Fourier transform infrared spectroscopy suggested a reduction of lignin and crystalline cellulose in OPEFB samples supplemented with CBH7B. Scanning electron microscopy revealed greater destruction extent of OPEFB strands in samples supplemented with CBH7B as compared with the nonsupplemented control. Therefore, CBH7B has the potential to complement commercial enzymes in hydrolyzing lignocellulosic biomass.
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http://dx.doi.org/10.1002/bab.1431DOI Listing
September 2016

Use of Megaprimer and Overlapping Extension PCR (OE-PCR) to Mutagenize and Enhance Cyclodextrin Glucosyltransferase (CGTase) Function.

Methods Mol Biol 2017 ;1498:385-396

Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.

Protein engineering is a very useful tool for probing structure-function relationships in proteins. Specifically, site-directed mutagenized proteins can provide useful insights into structural, binding and catalytic mechanisms of a protein, particularly when coupled with crystallization. In this chapter, we describe two protocols for performing site-directed mutagenesis of any protein-coding sequence, namely, megaprimer PCR and overlapping extension PCR (OE-PCR). We use as an example how these two SDM methods enhanced the function of a cyclodextrin glucosyltransferase (CGTase) from Bacillus lehensis strain G1.
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http://dx.doi.org/10.1007/978-1-4939-6472-7_27DOI Listing
January 2018

Structure Prediction of a Novel Exo-β-1,3-Glucanase: Insights into the Cold Adaptation of Psychrophilic Yeast Glaciozyma antarctica PI12.

Interdiscip Sci 2018 Mar 30;10(1):157-168. Epub 2016 Jul 30.

School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia.

We report a detailed structural analysis of the psychrophilic exo-β-1,3-glucanase (GaExg55) from Glaciozyma antarctica PI12. This study elucidates the structural basis of exo-1,3-β-1,3-glucanase from this psychrophilic yeast. The structural prediction of GaExg55 remains a challenge because of its low sequence identity (37 %). A 3D model was constructed for GaExg55. Threading approach was employed to determine a suitable template and generate optimal target-template alignment for establishing the model using MODELLER9v15. The primary sequence analysis of GaExg55 with other mesophilic exo-1,3-β-glucanases indicated that an increased flexibility conferred to the enzyme by a set of amino acids substitutions in the surface and loop regions of GaExg55, thereby facilitating its structure to cold adaptation. A comparison of GaExg55 with other mesophilic exo-β-1,3-glucanases proposed that the catalytic activity and structural flexibility at cold environment were attained through a reduced amount of hydrogen bonds and salt bridges, as well as an increased exposure of the hydrophobic side chains to the solvent. A molecular dynamics simulation was also performed using GROMACS software to evaluate the stability of the GaExg55 structure at varying low temperatures. The simulation result confirmed the above findings for cold adaptation of the psychrophilic GaExg55. Furthermore, the structural analysis of GaExg55 with large catalytic cleft and wide active site pocket confirmed the high activity of GaExg55 to hydrolyze polysaccharide substrates.
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http://dx.doi.org/10.1007/s12539-016-0180-9DOI Listing
March 2018

The role of alternative salt bridges in cold adaptation of a novel psychrophilic laminarinase.

J Biomol Struct Dyn 2017 06 5;35(8):1685-1692. Epub 2016 Aug 5.

d Faculty of Science and Technology, School of Biosciences and Biotechnology , Universiti Kebangsaan Malaysia , Kuala Lumpur , Malaysia.

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http://dx.doi.org/10.1080/07391102.2016.1191043DOI Listing
June 2017

Computational docking, molecular dynamics simulation and subsite structure analysis of a maltogenic amylase from Bacillus lehensis G1 provide insights into substrate and product specificity.

J Mol Graph Model 2016 06 23;67:1-13. Epub 2016 Apr 23.

Department of Bioprocess Engineering, Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, Skudai, Johor, Malaysia. Electronic address:

Maltogenic amylase (MAG1) from Bacillus lehensis G1 displayed the highest hydrolysis activity on β-cyclodextrin (β-CD) to produce maltose as a main product and exhibited high transglycosylation activity on malto-oligosaccharides with polymerization degree of three and above. These substrate and product specificities of MAG1 were elucidated from structural point of view in this study. A three-dimensional structure of MAG1 was constructed using homology modeling. Docking of β-CD and malto-oligosaccharides was then performed in the MAG1 active site. An aromatic platform in the active site was identified which is responsible in substrate recognition especially in determining the enzyme's preference toward β-CD. Molecular dynamics (MD) simulation showed MAG1 structure is most stable when docked with β-CD and least stable when docked with maltose. The docking analysis and MD simulation showed that the main subsites for substrate stabilization in the active site are -2, -1, +1 and +2. A bulky residue, Trp359 at the +2 subsite was identified to cause steric interference to the bound linear malto-oligosaccharides thus prevented it to occupy subsite +3, which can only be reached by a highly bent glucose molecule such as β-CD. The resulted modes of binding from docking simulation show a good correlation with the experimentally determined hydrolysis pattern. The subsite structure generated from this study led to a possible mode of action that revealed how maltose was mainly produced during hydrolysis. Furthermore, maltose only occupies subsite +1 and +2, therefore could not be hydrolyzed or transglycosylated by the enzyme. This important knowledge has paved the way for a novel structure-based molecular design for modulation of its catalytic activities.
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http://dx.doi.org/10.1016/j.jmgm.2016.04.004DOI Listing
June 2016
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