Kheng Low | Universiti Teknologi Malaysia - UTM (original) (raw)

Papers by Kheng Low

Research paper thumbnail of Escherichia coli extracellular secretion system an efficient protein production system for superior recovery of protein at reduced cost

Research paper thumbnail of Effect of temperature condition on xylanase production by immobilizing E. coli onto graphene oxide

The targeting of recombinant proteins for excretion into culture medium presents significant adva... more The targeting of recombinant proteins for excretion into culture medium presents significant advantages over cytoplasmic expression. However, during the excretion of recombinant protein, caution must be taken in order to avoid cell lysis due to pressure build-up through overproduction of the expressed recombinant protein in the periplasmic space. The recombinant E. coli cells were immobilized on the graphene oxide with temperature 30 degree celcius. The results presented showed that the immobilized cell is a promising method for high enzyme excretion and plasmid stability with less occurrences of cell lysis. The immobilized cell exhibited a 7% increase in the xylanase excretion 39% reduction of cell lysis compared with free cells.

Research paper thumbnail of Expression of xylanase on Escherichia coli using a truncated ice nucleation protein of Erwinia ananas (InaA)

Process Biochemistry, 2019

Cell surface display involves the anchoring of enzymes on the surface of cells to be used as whol... more Cell surface display involves the anchoring of enzymes on the surface of cells to be used as whole-cell biocatalysts. In this study, an ice nucleation protein (INP) anchor from Erwinia ananas IN-10, InaA, was used to display xylanase. The ability of InaA to be targeted to the outer membrane was compared to cells displaying xylanase using two other INPs, InaK and InaZ. SDS-PAGE and western blot of the outer membrane fraction proved that surface targeting was successful. Whole-cell xylanase activity showed that InaA anchored xylanase gave an activity of 92.2 U/g dry cell weight which was up to three times higher than the other two display constructs used. Surface anchoring of InaA was up to four times better compared to the other two INP anchors as was confirmed by flow cytometry. Expression of InaA on the surface was optimized by one-factor-at-a-time (OFAT) to obtain the optimum parameter conditions for highest surface expression. The results presented showed that InaA is an excellent INP for surface display for xylanase and has great potential in the degradation of xylan.

Research paper thumbnail of Optimization of a Heterologous Signal Peptide by Site-Directed Mutagenesis for Improved Secretion of Recombinant Proteins in Escherichia coli

Microbial Physiology, 2012

A heterologous signal peptide (SP) from Bacillus sp. G1 was optimized for secretion of recombinan... more A heterologous signal peptide (SP) from Bacillus sp. G1 was optimized for secretion of recombinant cyclodextrin glucanotransferase (CGTase) to the periplasmic and, eventually, extracellular space of Escherichia coli. Eight mutant SPs were constructed using site-directed mutagenesis to improve the secretion of recombinant CGTase. M5 is a mutated SP in which replacement of an isoleucine residue in the h-region to glycine created a helix-breaking or G-turn motif with decreased hydrophobicity. The mutant SP resulted in 110 and 94% increases in periplasmic and extracellular recombinant CGTase, respectively, compared to the wild-type SP at a similar level of cell lysis. The formation of intracellular inclusion bodies was also reduced, as determined by sodium dodecyl sulfate-polyacrylamyde gel electrophoresis, when this mutated SP was used. The addition of as low as 0.08% glycine at the beginning of cell growth improved cell viability of the E. coli host. Secretory production of other prot...

Research paper thumbnail of Enhanced secretory production of hemolysin-mediated cyclodextrin glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system

Journal of Biotechnology, 2010

The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferas... more The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferase (CGTase) into the extracellular medium when it was fused to the Cterminal 61 amino acids of HlyA (HlyAs(61)). To produce an improved-secretion variant, the hly components (hlyAs, hlyB and hlyD) were engineered by directed evolution using errorprone PCR. Hly mutants were screened on solid LB-starch plate for halo zone larger than the parent strain. Through screening of about 1 × 10(4) Escherichia coli BL21(DE3) transformants, we succeeded in isolating five mutants that showed a 35-217% increase in the secretion level of CGTase-HlyAs(61) relative to the wild-type strain. The mutation sites of each mutant were located at HlyB, primarily along the transmembrane domain, implying that the corresponding region was important for the improved secretion of the target protein. In this study we describe the finding of novel site(s) of HlyB responsible for enhancing secretion of CGTase in E. coli.

Research paper thumbnail of An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production

Journal of Industrial Microbiology & Biotechnology, 2011

Direct transport of recombinant protein from cytosol to extracellular medium offers great advanta... more Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage.

Research paper thumbnail of Draft Genome Sequence of Cellulomonas sp. PS-H5, Isolated from Sekinchan Beach in Selangor, Malaysia

Microbiology Resource Announcements

Cellulomonas sp. strain PS-H5 was isolated from Sekinchan Beach in Selangor, Malaysia, using an e... more Cellulomonas sp. strain PS-H5 was isolated from Sekinchan Beach in Selangor, Malaysia, using an ex situ cultivation method. The present work reports a high-quality draft annotated genome sequence of this strain and suggests its potential glycoside hydrolase enzymes for cellulose, hemicellulose, and starch degradations.

Research paper thumbnail of Carbon nanomaterial properties help to enhance xylanase production from recombinant Kluyveromyces lactis through a cell immobilization method

Applied Microbiology and Biotechnology

Research paper thumbnail of Emergence of nanomaterials as potential immobilization supports for whole cell biocatalysts and cell toxicity effects

Biotechnology and Applied Biochemistry

Research paper thumbnail of An improvement in fermentability of acid-hydrolysed hemicellulose from kenaf stem for xylitol production

International Journal of Food Engineering

In recent years, there has been a growing interest in the use of agricultural biomass for ferment... more In recent years, there has been a growing interest in the use of agricultural biomass for fermentation purposes; however, efficient strategies to counter lignocellulose inhibition are warranted to enhance xylitol production performance. Dilute-acid hydrolysis has been studied to selectively release a significant portion of xylose from hemicellulose, while leaving cellulose and lignin intact. The formation of inhibitory compounds, however, could jeopardise the overall performance during fermentation to produce xylitol. In this study, the fermentability of nitric acid-hydrolysed kenaf stem was substantially improved, through either adaptive evolution of the recombinant Escherichia coli BL21 (DE3) or removal of fermentation inhibitors by detoxification with activated carbon. Both methods were compared to evaluate the superiority in fermentative performance. In the fermentation with detoxified hemicellulosic hydrolysate, the non-adapted strain produced the highest xylitol concentration ...

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

Research paper thumbnail of Chitinase and chitoologosaccharide production by trichoderma oblongisporium bisset using pretreated shrimp waste

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

Scientific Reports

Kenaf (Hibiscus cannabinus L.), a potential fibre crop with a desirably high growth rate, could s... more 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 3 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. Xylitol is a non-fermentable pentahydroxy sugar-alcohol found in some fruits and vegetables including mushrooms, lettuces, berries and corns 1,2. The U.S. Food and Drug Administration (FDA) has considered xylitol as a healthy sugar substitute, given its comparable sweetness as that of regular sucrose, coupled with a 40% lower calorific value 3. The rising public-health concerns with sugar (i.e. sucrose) have spurred the global demand for xylitol owing to its anticarcinogenicity and prebiotic nature 4. The global production of xylitol has been reported to reach 160,000 tonnes with an estimated selling price of US$ 4-5 per kg 5. The market value of xylitol was worth approximately US$ 670 million in 2013, with the Asia-Pacific region being the important player 6. Thus, xylitol has been classified as a potential value-added chemicals from bio-renewable resources, according to the U.S. Department of Energy 2. The chemical production of xylitol involves catalytic hydrogenation of a highly purified D-xylose in the existence of Raney nickel catalysts 7. The extreme pressures and temperatures entailed by the chemical production translate, in practice, into considerable consumption of energy and cost 8. On the contrary, the biochemical approach through microbial cultivation constitutes a viable alternative to the chemical counterpart in the production of xylitol, owing to its lesser complexity, inherent sustainability and environmentally-friendly nature 7 .

Research paper thumbnail of An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production

Journal of Industrial Microbiology & Biotechnology, 2011

Direct transport of recombinant protein from cytosol to extracellular medium offers great advanta... more Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 lM IPTG, 1.0% (w/v) arabinose and 34.7°C postinduction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage.

Research paper thumbnail of A comparative genomic analysis of the alkalitolerant soil bacterium Bacillus lehensis G1

Gene, 2014

Bacillus lehensis G1 is a Gram-positive, moderately alkalitolerant bacterium isolated from soil s... more Bacillus lehensis G1 is a Gram-positive, moderately alkalitolerant bacterium isolated from soil samples. B. lehensis produces cyclodextrin glucanotransferase (CGTase), an enzyme that has enabled the extensive use of cyclodextrin in foodstuffs, chemicals, and pharmaceuticals. The genome sequence of B. lehensis G1 consists of a single circular 3.99 Mb chromosome containing 4017 protein-coding sequences (CDSs), of which 2818 (70.15%) have assigned biological roles, 936 (23.30%) have conserved domains with unknown functions, and 263 (6.55%) have no match with any protein database. Bacillus clausii KSM-K16 was established as the closest relative to B. lehensis G1 based on gene content similarity and 16S rRNA phylogenetic analysis. A total of 2820 proteins from B. lehensis G1 were found to have orthologues in B. clausii, including sodium-proton antiporters, transport proteins, and proteins involved in ATP synthesis. A comparative analysis of these proteins and those in B. clausii and other alkaliphilic Bacillus species was carried out to investigate their contributions towards the alkalitolerance of the microorganism. The similarities and differences in alkalitolerance-related genes among alkalitolerant/alkaliphilic Bacillus species highlight the complex mechanism of pH homeostasis. The B. lehensis G1 genome was also mined for proteins and enzymes with potential viability for industrial and commercial purposes.

Research paper thumbnail of Heterologous Expression of Proteins from Cold-Adapted Yeasts in Suitable Hosts: Methods and Applications

Cold-adapted Yeasts, 2013

Research paper thumbnail of Optimization of a Bacillus sp signal peptide for improved recombinant protein secretion and cell viability in Escherichia coli

Research paper thumbnail of An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production

Journal of Industrial Microbiology & Biotechnology, 2011

Direct transport of recombinant protein from cytosol to extracellular medium offers great advanta... more Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 lM IPTG, 1.0% (w/v) arabinose and 34.7°C postinduction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage.

Research paper thumbnail of Enhanced secretory production of hemolysin-mediated cyclodextrin glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system

Journal of Biotechnology, 2010

The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferas... more The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferase (CGTase) into the extracellular medium when it was fused to the C-terminal 61 amino acids of HlyA (HlyAs 61 ). To produce an improved-secretion variant, the hly components (hlyAs, hlyB and hlyD) were engineered by directed evolution using error-prone PCR. Hly mutants were screened on solid LB-starch plate for halo zone larger than the parent strain. Through screening of about 1 × 10 4 Escherichia coli BL21(DE3) transformants, we succeeded in isolating five mutants that showed a 35-217% increase in the secretion level of CGTase-HlyAs 61 relative to the wild-type strain. The mutation sites of each mutant were located at HlyB, primarily along the transmembrane domain, implying that the corresponding region was important for the improved secretion of the target protein. In this study we describe the finding of novel site(s) of HlyB responsible for enhancing secretion of CGTase in E. coli.

Research paper thumbnail of Optimisation of signal peptide for recombinant protein secretion in bacterial hosts

Applied Microbiology and Biotechnology, 2013

Escherichia coli-the powerhouse for recombinant protein production-is rapidly gaining status as a... more Escherichia coli-the powerhouse for recombinant protein production-is rapidly gaining status as a reliable and efficient host for secretory expression. An improved understanding of protein translocation processes and its mechanisms has inspired and accelerated the development of new tools and applications in this field and, in particular, a more efficient secretion signal. Several important characteristics and requirements are summarised for the design of a more efficient signal peptide for the production of recombinant proteins in E. coli. General approaches and strategies to optimise the signal peptide, including the selection and modification of the signal peptide components, are included. Several challenges in the secretory production of recombinant proteins are discussed, and research approaches designed to meet these challenges are proposed.

Research paper thumbnail of Escherichia coli extracellular secretion system an efficient protein production system for superior recovery of protein at reduced cost

Research paper thumbnail of Effect of temperature condition on xylanase production by immobilizing E. coli onto graphene oxide

The targeting of recombinant proteins for excretion into culture medium presents significant adva... more The targeting of recombinant proteins for excretion into culture medium presents significant advantages over cytoplasmic expression. However, during the excretion of recombinant protein, caution must be taken in order to avoid cell lysis due to pressure build-up through overproduction of the expressed recombinant protein in the periplasmic space. The recombinant E. coli cells were immobilized on the graphene oxide with temperature 30 degree celcius. The results presented showed that the immobilized cell is a promising method for high enzyme excretion and plasmid stability with less occurrences of cell lysis. The immobilized cell exhibited a 7% increase in the xylanase excretion 39% reduction of cell lysis compared with free cells.

Research paper thumbnail of Expression of xylanase on Escherichia coli using a truncated ice nucleation protein of Erwinia ananas (InaA)

Process Biochemistry, 2019

Cell surface display involves the anchoring of enzymes on the surface of cells to be used as whol... more Cell surface display involves the anchoring of enzymes on the surface of cells to be used as whole-cell biocatalysts. In this study, an ice nucleation protein (INP) anchor from Erwinia ananas IN-10, InaA, was used to display xylanase. The ability of InaA to be targeted to the outer membrane was compared to cells displaying xylanase using two other INPs, InaK and InaZ. SDS-PAGE and western blot of the outer membrane fraction proved that surface targeting was successful. Whole-cell xylanase activity showed that InaA anchored xylanase gave an activity of 92.2 U/g dry cell weight which was up to three times higher than the other two display constructs used. Surface anchoring of InaA was up to four times better compared to the other two INP anchors as was confirmed by flow cytometry. Expression of InaA on the surface was optimized by one-factor-at-a-time (OFAT) to obtain the optimum parameter conditions for highest surface expression. The results presented showed that InaA is an excellent INP for surface display for xylanase and has great potential in the degradation of xylan.

Research paper thumbnail of Optimization of a Heterologous Signal Peptide by Site-Directed Mutagenesis for Improved Secretion of Recombinant Proteins in Escherichia coli

Microbial Physiology, 2012

A heterologous signal peptide (SP) from Bacillus sp. G1 was optimized for secretion of recombinan... more A heterologous signal peptide (SP) from Bacillus sp. G1 was optimized for secretion of recombinant cyclodextrin glucanotransferase (CGTase) to the periplasmic and, eventually, extracellular space of Escherichia coli. Eight mutant SPs were constructed using site-directed mutagenesis to improve the secretion of recombinant CGTase. M5 is a mutated SP in which replacement of an isoleucine residue in the h-region to glycine created a helix-breaking or G-turn motif with decreased hydrophobicity. The mutant SP resulted in 110 and 94% increases in periplasmic and extracellular recombinant CGTase, respectively, compared to the wild-type SP at a similar level of cell lysis. The formation of intracellular inclusion bodies was also reduced, as determined by sodium dodecyl sulfate-polyacrylamyde gel electrophoresis, when this mutated SP was used. The addition of as low as 0.08% glycine at the beginning of cell growth improved cell viability of the E. coli host. Secretory production of other prot...

Research paper thumbnail of Enhanced secretory production of hemolysin-mediated cyclodextrin glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system

Journal of Biotechnology, 2010

The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferas... more The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferase (CGTase) into the extracellular medium when it was fused to the Cterminal 61 amino acids of HlyA (HlyAs(61)). To produce an improved-secretion variant, the hly components (hlyAs, hlyB and hlyD) were engineered by directed evolution using errorprone PCR. Hly mutants were screened on solid LB-starch plate for halo zone larger than the parent strain. Through screening of about 1 × 10(4) Escherichia coli BL21(DE3) transformants, we succeeded in isolating five mutants that showed a 35-217% increase in the secretion level of CGTase-HlyAs(61) relative to the wild-type strain. The mutation sites of each mutant were located at HlyB, primarily along the transmembrane domain, implying that the corresponding region was important for the improved secretion of the target protein. In this study we describe the finding of novel site(s) of HlyB responsible for enhancing secretion of CGTase in E. coli.

Research paper thumbnail of An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production

Journal of Industrial Microbiology & Biotechnology, 2011

Direct transport of recombinant protein from cytosol to extracellular medium offers great advanta... more Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage.

Research paper thumbnail of Draft Genome Sequence of Cellulomonas sp. PS-H5, Isolated from Sekinchan Beach in Selangor, Malaysia

Microbiology Resource Announcements

Cellulomonas sp. strain PS-H5 was isolated from Sekinchan Beach in Selangor, Malaysia, using an e... more Cellulomonas sp. strain PS-H5 was isolated from Sekinchan Beach in Selangor, Malaysia, using an ex situ cultivation method. The present work reports a high-quality draft annotated genome sequence of this strain and suggests its potential glycoside hydrolase enzymes for cellulose, hemicellulose, and starch degradations.

Research paper thumbnail of Carbon nanomaterial properties help to enhance xylanase production from recombinant Kluyveromyces lactis through a cell immobilization method

Applied Microbiology and Biotechnology

Research paper thumbnail of Emergence of nanomaterials as potential immobilization supports for whole cell biocatalysts and cell toxicity effects

Biotechnology and Applied Biochemistry

Research paper thumbnail of An improvement in fermentability of acid-hydrolysed hemicellulose from kenaf stem for xylitol production

International Journal of Food Engineering

In recent years, there has been a growing interest in the use of agricultural biomass for ferment... more In recent years, there has been a growing interest in the use of agricultural biomass for fermentation purposes; however, efficient strategies to counter lignocellulose inhibition are warranted to enhance xylitol production performance. Dilute-acid hydrolysis has been studied to selectively release a significant portion of xylose from hemicellulose, while leaving cellulose and lignin intact. The formation of inhibitory compounds, however, could jeopardise the overall performance during fermentation to produce xylitol. In this study, the fermentability of nitric acid-hydrolysed kenaf stem was substantially improved, through either adaptive evolution of the recombinant Escherichia coli BL21 (DE3) or removal of fermentation inhibitors by detoxification with activated carbon. Both methods were compared to evaluate the superiority in fermentative performance. In the fermentation with detoxified hemicellulosic hydrolysate, the non-adapted strain produced the highest xylitol concentration ...

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

Research paper thumbnail of Chitinase and chitoologosaccharide production by trichoderma oblongisporium bisset using pretreated shrimp waste

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

Scientific Reports

Kenaf (Hibiscus cannabinus L.), a potential fibre crop with a desirably high growth rate, could s... more 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 3 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. Xylitol is a non-fermentable pentahydroxy sugar-alcohol found in some fruits and vegetables including mushrooms, lettuces, berries and corns 1,2. The U.S. Food and Drug Administration (FDA) has considered xylitol as a healthy sugar substitute, given its comparable sweetness as that of regular sucrose, coupled with a 40% lower calorific value 3. The rising public-health concerns with sugar (i.e. sucrose) have spurred the global demand for xylitol owing to its anticarcinogenicity and prebiotic nature 4. The global production of xylitol has been reported to reach 160,000 tonnes with an estimated selling price of US$ 4-5 per kg 5. The market value of xylitol was worth approximately US$ 670 million in 2013, with the Asia-Pacific region being the important player 6. Thus, xylitol has been classified as a potential value-added chemicals from bio-renewable resources, according to the U.S. Department of Energy 2. The chemical production of xylitol involves catalytic hydrogenation of a highly purified D-xylose in the existence of Raney nickel catalysts 7. The extreme pressures and temperatures entailed by the chemical production translate, in practice, into considerable consumption of energy and cost 8. On the contrary, the biochemical approach through microbial cultivation constitutes a viable alternative to the chemical counterpart in the production of xylitol, owing to its lesser complexity, inherent sustainability and environmentally-friendly nature 7 .

Research paper thumbnail of An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production

Journal of Industrial Microbiology & Biotechnology, 2011

Direct transport of recombinant protein from cytosol to extracellular medium offers great advanta... more Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 lM IPTG, 1.0% (w/v) arabinose and 34.7°C postinduction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage.

Research paper thumbnail of A comparative genomic analysis of the alkalitolerant soil bacterium Bacillus lehensis G1

Gene, 2014

Bacillus lehensis G1 is a Gram-positive, moderately alkalitolerant bacterium isolated from soil s... more Bacillus lehensis G1 is a Gram-positive, moderately alkalitolerant bacterium isolated from soil samples. B. lehensis produces cyclodextrin glucanotransferase (CGTase), an enzyme that has enabled the extensive use of cyclodextrin in foodstuffs, chemicals, and pharmaceuticals. The genome sequence of B. lehensis G1 consists of a single circular 3.99 Mb chromosome containing 4017 protein-coding sequences (CDSs), of which 2818 (70.15%) have assigned biological roles, 936 (23.30%) have conserved domains with unknown functions, and 263 (6.55%) have no match with any protein database. Bacillus clausii KSM-K16 was established as the closest relative to B. lehensis G1 based on gene content similarity and 16S rRNA phylogenetic analysis. A total of 2820 proteins from B. lehensis G1 were found to have orthologues in B. clausii, including sodium-proton antiporters, transport proteins, and proteins involved in ATP synthesis. A comparative analysis of these proteins and those in B. clausii and other alkaliphilic Bacillus species was carried out to investigate their contributions towards the alkalitolerance of the microorganism. The similarities and differences in alkalitolerance-related genes among alkalitolerant/alkaliphilic Bacillus species highlight the complex mechanism of pH homeostasis. The B. lehensis G1 genome was also mined for proteins and enzymes with potential viability for industrial and commercial purposes.

Research paper thumbnail of Heterologous Expression of Proteins from Cold-Adapted Yeasts in Suitable Hosts: Methods and Applications

Cold-adapted Yeasts, 2013

Research paper thumbnail of Optimization of a Bacillus sp signal peptide for improved recombinant protein secretion and cell viability in Escherichia coli

Research paper thumbnail of An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production

Journal of Industrial Microbiology & Biotechnology, 2011

Direct transport of recombinant protein from cytosol to extracellular medium offers great advanta... more Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 lM IPTG, 1.0% (w/v) arabinose and 34.7°C postinduction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage.

Research paper thumbnail of Enhanced secretory production of hemolysin-mediated cyclodextrin glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system

Journal of Biotechnology, 2010

The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferas... more The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferase (CGTase) into the extracellular medium when it was fused to the C-terminal 61 amino acids of HlyA (HlyAs 61 ). To produce an improved-secretion variant, the hly components (hlyAs, hlyB and hlyD) were engineered by directed evolution using error-prone PCR. Hly mutants were screened on solid LB-starch plate for halo zone larger than the parent strain. Through screening of about 1 × 10 4 Escherichia coli BL21(DE3) transformants, we succeeded in isolating five mutants that showed a 35-217% increase in the secretion level of CGTase-HlyAs 61 relative to the wild-type strain. The mutation sites of each mutant were located at HlyB, primarily along the transmembrane domain, implying that the corresponding region was important for the improved secretion of the target protein. In this study we describe the finding of novel site(s) of HlyB responsible for enhancing secretion of CGTase in E. coli.

Research paper thumbnail of Optimisation of signal peptide for recombinant protein secretion in bacterial hosts

Applied Microbiology and Biotechnology, 2013

Escherichia coli-the powerhouse for recombinant protein production-is rapidly gaining status as a... more Escherichia coli-the powerhouse for recombinant protein production-is rapidly gaining status as a reliable and efficient host for secretory expression. An improved understanding of protein translocation processes and its mechanisms has inspired and accelerated the development of new tools and applications in this field and, in particular, a more efficient secretion signal. Several important characteristics and requirements are summarised for the design of a more efficient signal peptide for the production of recombinant proteins in E. coli. General approaches and strategies to optimise the signal peptide, including the selection and modification of the signal peptide components, are included. Several challenges in the secretory production of recombinant proteins are discussed, and research approaches designed to meet these challenges are proposed.