SAJJAD AHMAD | University of Sargodha (original) (raw)

Papers by SAJJAD AHMAD

BioMed Research International, 2015

This study describes a simplified approach for enhanced expression and secretion of a pharmaceuti... more This study describes a simplified approach for enhanced expression and secretion of a pharmaceutically important human cytokine, that is, granulocyte colony stimulating factor (GCSF), in the culture supernatant ofBacillus subtilisSCK6 cells. Codon optimized GCSF and pNWPH vector containing SpymwC signal sequence were amplified by prolonged overlap extension PCR to generate multimeric plasmid DNA, which was used directly to transformB. subtilisSCK6 supercompetent cells. Expression ofGCSFwas monitored in the culture supernatant for 120 hours. The highest expression, which corresponded to 17% of the total secretory protein, was observed at 72 hours of growth. Following ammonium sulphate precipitation, GCSF was purified to near homogeneity by fast protein liquid chromatography on a QFF anion exchange column. Circular dichroism spectroscopic analysis showed that the secondary structure contents of the purified GCSF are similar to the commercially available GCSF. Biological activity, as r...

World Journal of Microbiology and Biotechnology, 2015

The sequence and structure of mRNA plays an important role in solubility and expression of the tr... more The sequence and structure of mRNA plays an important role in solubility and expression of the translated protein. To divulge the role of mRNA secondary structure and its thermodynamics in the expression level of the recombinant endoglucanase in Escherichia coli, 5'-end of the mRNA was thermodynamically optimized. Molecular engineering was done by introducing two silent synonymous mutations at positions +5 (UCU with UCC) and +7 (UUC with UUU) of the…

The sequence and structure of mRNA plays an important role in solubility and expression of the tr... more The sequence and structure of mRNA plays an important role in solubility and expression of the translated protein. To divulge the role of mRNA secondary structure and its thermodynamics in the expression level of the recombinant endoglucanase in Escherichia coli, 5′-end of the mRNA was thermodynamically optimized. Molecular engineering was done by introducing two silent synonymous mutations at positions +5 (UCU with UCC) and +7 (UUC with UUU) of the 5′-end of mRNA to relieve hybridization with ribosomal binding site. Two variants of glycoside hydrolase family six endoglucanase, wild type (cel6A.wt) and mutant (cel6A.mut) from Thermobifida fusca were expressed and characterized in E. coli using T7 promoter-based expression vector; pET22b(+). Enhanced expression level of engineered construct (Cel6A.mut) with ∆G = −2.7 kcal mol−1was observed. It showed up to ~45 % higher expression as compared to the wild type construct (Cel6A.wt) having ∆G = −7.8 kcal mol−1 and ~25 % expression to the total cell proteins. Heterologous protein was purified by heating the recombinant E. coli BL21 (DE3) CodonPlus at 60 °C. The optimum pH for enzyme activity was six and optimum temperature was 60 °C. Maximum activity was observed 4.5 Umg−1 on CMC. Hydrolytic activity was also observed on insoluble substrates, i.e. RAC (2.8 Umg−1), alkali treated bagass (1.7 Umg−1), filter paper (1.2 Umg−1) and BMCC (0.3 Umg−1). Metal ions affect endoglucanase activity in different ways. Only Fe2+ exhibited 20.8 % stimulatory effects on enzyme activity. Enzyme activity was profoundly inhibited by Hg2+ (91.8 %).

Increasing specific activity of cellulase on solid cellulosic materials would be among the top pr... more Increasing specific activity of cellulase on solid cellulosic materials would be among the top priorities for second-generation biorefineries. However, the complicated relationship among the heterogeneity of solid cellulosic materials and different action mode cellulase components results in great challenges in cellulase engineering. We applied directed evolution to a Clostridium phytofermentans ISDg glycoside hydrolase family 9 processive endoglucanase (CpCel9) for enhanced hydrolytic performance by using Bacillus subtilis as a host for cloning and expression. Several CpCel9 mutants with both increased expression level and enhanced specific activity on the solid cellulosic material were obtained. The most active mutant, which also exhibits an increased expression level, had more than threefold specific activity than that of wild type on regenerated amorphous cellulose. Most mutation sites were located in the family 3 cellulose-binding module near to its catalytic module, which might guide the entrance of glucan into the catalytic module. This study suggested that directed evolution by combining B. subtilis secretory protein expression host and solid cellulosic substrates would be a powerful tool to evolve more active cellulase mutants for cost-effective biosaccharification process.

Journal of biotechnology, 2012

Journal of biotechnology, 2010

Clostridium thermocellum encodes a xylanase gene (xynC) which is the major component of its cellu... more Clostridium thermocellum encodes a xylanase gene (xynC) which is the major component of its cellulosome. XynC is a multidomain enzyme comprising of a substrate binding domain at the N-terminal followed by the catalytic domain and a dockerin domain. To study the influence of binding domain on activity, stability and expression of the enzyme the protein with the binding domain at C-terminal (XynC-CB), and the one with the binding domain at both N-and C-terminal (XynC-BCB) were expressed in E. coli. Recombinant plasmids, pXynC-CB and pXynC-BCB were constructed by inserting the corresponding gene in pET22b(+). XynC-CB and XynC-BCB were expressed at levels around 30% and 33% of the total E. coli cell proteins, respectively, while losing 40% and 20% of their activities at 70 • C for 120 min, respectively. The specific activities of XynC-CB , XynC-BCB were 76 and 98 U mg −1 , while the activities on equimolar basis were 4410 and 7450 U M −1 against birchwood xylan, respectively. Their overall activities produced in the culture were 3660 and 5430 U L −1 OD 600 −1 . Substrate binding studies showed that in case of XynC-C 51% of the activity remained unbound to birchwood xylan, whereas in the cases of XynC-BC, XynC-CB and XynC-BCB the activities left unbound were 33%, 32% and 12%, respectively, under the assay conditions used. Similar binding values were obtained in the case of oat spelt xylan. K m values for XynC-CB and XynC-BCB against birchwood xylan were found to be 3.1 and 1.47 mg ml −1 , respectively. Thus addition of a second carbohydrate binding domain at the C-terminal of the catalytic domain enhances activity, substrate affinity as well as thermostability.

Journal of Biotechnology, 2010

Two major xylanase components, XynC and XynZ from the anaerobic thermophilic bacterium, Clostridi... more Two major xylanase components, XynC and XynZ from the anaerobic thermophilic bacterium, Clostridium thermocellum cellulosome were cloned and expressed with and without non-catalytic domains in E. coli. Two constructs of XynC, one with its cellulose binding domain and the catalytic domain (pXynC-BC) and the other with only the catalytic domain (pXynC-C) were produced. For XynZ the constructs produced were pXynZ-BDC, which included the dockerin domain, and pXynZ-C, which did not. E. coli cells transformed with pXynC-BC or pXynZ-BDC gave xylanase expression of 30% and 25% total cell proteins, respectively. Transformation of E. coli cells with the constructs carrying only the catalytic domains gave expression levels of ∼45% in each case. The specific activities of XynC with and without the non-catalytic domains were similar, but for XynZ the specific activity of the enzyme without the non-catalytic domains was ∼5-fold greater than that of the intact enzyme. The total activity increased from 1925 U l−1 OD600−1 for XynC-BC to 3050 U l−1 OD600−1 for XynC-C. However, the overall increase in activity was ∼9-fold higher for XynZ-C (32,900 U l−1 OD600−1) versus XynZ-BDC (3665 U l−1 OD600−1). Both the enzymes with and without non-catalytic domains were found to be quite stable over a broad pH range (pH 4–9). XynZ-C was more thermostable than XynZ-BDC as it retained 87% of xylanase activity when incubated at 70 °C for 2 h as compared to 42% for XynZ-BDC. However, XynC-BC retained 70% activity on incubation at 70 °C for 2 h but XynC-C lost all activity under the same conditions. Km values for XynC-BC and XynC-C determined on soluble xylan were 3.1 and 3.6 mg ml−1, respectively, whereas these values for XynZ-BDC and XynZ-C were 33.3 and 15.4 mg ml−1, respectively. Thus the production of xylanase activity by expressing only the catalytic domains of XynC and XynZ is significantly enhanced.

BioMed Research International, 2015

This study describes a simplified approach for enhanced expression and secretion of a pharmaceuti... more This study describes a simplified approach for enhanced expression and secretion of a pharmaceutically important human cytokine, that is, granulocyte colony stimulating factor (GCSF), in the culture supernatant ofBacillus subtilisSCK6 cells. Codon optimized GCSF and pNWPH vector containing SpymwC signal sequence were amplified by prolonged overlap extension PCR to generate multimeric plasmid DNA, which was used directly to transformB. subtilisSCK6 supercompetent cells. Expression ofGCSFwas monitored in the culture supernatant for 120 hours. The highest expression, which corresponded to 17% of the total secretory protein, was observed at 72 hours of growth. Following ammonium sulphate precipitation, GCSF was purified to near homogeneity by fast protein liquid chromatography on a QFF anion exchange column. Circular dichroism spectroscopic analysis showed that the secondary structure contents of the purified GCSF are similar to the commercially available GCSF. Biological activity, as r...

World Journal of Microbiology and Biotechnology, 2015

The sequence and structure of mRNA plays an important role in solubility and expression of the tr... more The sequence and structure of mRNA plays an important role in solubility and expression of the translated protein. To divulge the role of mRNA secondary structure and its thermodynamics in the expression level of the recombinant endoglucanase in Escherichia coli, 5'-end of the mRNA was thermodynamically optimized. Molecular engineering was done by introducing two silent synonymous mutations at positions +5 (UCU with UCC) and +7 (UUC with UUU) of the…

The sequence and structure of mRNA plays an important role in solubility and expression of the tr... more The sequence and structure of mRNA plays an important role in solubility and expression of the translated protein. To divulge the role of mRNA secondary structure and its thermodynamics in the expression level of the recombinant endoglucanase in Escherichia coli, 5′-end of the mRNA was thermodynamically optimized. Molecular engineering was done by introducing two silent synonymous mutations at positions +5 (UCU with UCC) and +7 (UUC with UUU) of the 5′-end of mRNA to relieve hybridization with ribosomal binding site. Two variants of glycoside hydrolase family six endoglucanase, wild type (cel6A.wt) and mutant (cel6A.mut) from Thermobifida fusca were expressed and characterized in E. coli using T7 promoter-based expression vector; pET22b(+). Enhanced expression level of engineered construct (Cel6A.mut) with ∆G = −2.7 kcal mol−1was observed. It showed up to ~45 % higher expression as compared to the wild type construct (Cel6A.wt) having ∆G = −7.8 kcal mol−1 and ~25 % expression to the total cell proteins. Heterologous protein was purified by heating the recombinant E. coli BL21 (DE3) CodonPlus at 60 °C. The optimum pH for enzyme activity was six and optimum temperature was 60 °C. Maximum activity was observed 4.5 Umg−1 on CMC. Hydrolytic activity was also observed on insoluble substrates, i.e. RAC (2.8 Umg−1), alkali treated bagass (1.7 Umg−1), filter paper (1.2 Umg−1) and BMCC (0.3 Umg−1). Metal ions affect endoglucanase activity in different ways. Only Fe2+ exhibited 20.8 % stimulatory effects on enzyme activity. Enzyme activity was profoundly inhibited by Hg2+ (91.8 %).

Increasing specific activity of cellulase on solid cellulosic materials would be among the top pr... more Increasing specific activity of cellulase on solid cellulosic materials would be among the top priorities for second-generation biorefineries. However, the complicated relationship among the heterogeneity of solid cellulosic materials and different action mode cellulase components results in great challenges in cellulase engineering. We applied directed evolution to a Clostridium phytofermentans ISDg glycoside hydrolase family 9 processive endoglucanase (CpCel9) for enhanced hydrolytic performance by using Bacillus subtilis as a host for cloning and expression. Several CpCel9 mutants with both increased expression level and enhanced specific activity on the solid cellulosic material were obtained. The most active mutant, which also exhibits an increased expression level, had more than threefold specific activity than that of wild type on regenerated amorphous cellulose. Most mutation sites were located in the family 3 cellulose-binding module near to its catalytic module, which might guide the entrance of glucan into the catalytic module. This study suggested that directed evolution by combining B. subtilis secretory protein expression host and solid cellulosic substrates would be a powerful tool to evolve more active cellulase mutants for cost-effective biosaccharification process.

Journal of biotechnology, 2012

Journal of biotechnology, 2010

Clostridium thermocellum encodes a xylanase gene (xynC) which is the major component of its cellu... more Clostridium thermocellum encodes a xylanase gene (xynC) which is the major component of its cellulosome. XynC is a multidomain enzyme comprising of a substrate binding domain at the N-terminal followed by the catalytic domain and a dockerin domain. To study the influence of binding domain on activity, stability and expression of the enzyme the protein with the binding domain at C-terminal (XynC-CB), and the one with the binding domain at both N-and C-terminal (XynC-BCB) were expressed in E. coli. Recombinant plasmids, pXynC-CB and pXynC-BCB were constructed by inserting the corresponding gene in pET22b(+). XynC-CB and XynC-BCB were expressed at levels around 30% and 33% of the total E. coli cell proteins, respectively, while losing 40% and 20% of their activities at 70 • C for 120 min, respectively. The specific activities of XynC-CB , XynC-BCB were 76 and 98 U mg −1 , while the activities on equimolar basis were 4410 and 7450 U M −1 against birchwood xylan, respectively. Their overall activities produced in the culture were 3660 and 5430 U L −1 OD 600 −1 . Substrate binding studies showed that in case of XynC-C 51% of the activity remained unbound to birchwood xylan, whereas in the cases of XynC-BC, XynC-CB and XynC-BCB the activities left unbound were 33%, 32% and 12%, respectively, under the assay conditions used. Similar binding values were obtained in the case of oat spelt xylan. K m values for XynC-CB and XynC-BCB against birchwood xylan were found to be 3.1 and 1.47 mg ml −1 , respectively. Thus addition of a second carbohydrate binding domain at the C-terminal of the catalytic domain enhances activity, substrate affinity as well as thermostability.

Journal of Biotechnology, 2010

Two major xylanase components, XynC and XynZ from the anaerobic thermophilic bacterium, Clostridi... more Two major xylanase components, XynC and XynZ from the anaerobic thermophilic bacterium, Clostridium thermocellum cellulosome were cloned and expressed with and without non-catalytic domains in E. coli. Two constructs of XynC, one with its cellulose binding domain and the catalytic domain (pXynC-BC) and the other with only the catalytic domain (pXynC-C) were produced. For XynZ the constructs produced were pXynZ-BDC, which included the dockerin domain, and pXynZ-C, which did not. E. coli cells transformed with pXynC-BC or pXynZ-BDC gave xylanase expression of 30% and 25% total cell proteins, respectively. Transformation of E. coli cells with the constructs carrying only the catalytic domains gave expression levels of ∼45% in each case. The specific activities of XynC with and without the non-catalytic domains were similar, but for XynZ the specific activity of the enzyme without the non-catalytic domains was ∼5-fold greater than that of the intact enzyme. The total activity increased from 1925 U l−1 OD600−1 for XynC-BC to 3050 U l−1 OD600−1 for XynC-C. However, the overall increase in activity was ∼9-fold higher for XynZ-C (32,900 U l−1 OD600−1) versus XynZ-BDC (3665 U l−1 OD600−1). Both the enzymes with and without non-catalytic domains were found to be quite stable over a broad pH range (pH 4–9). XynZ-C was more thermostable than XynZ-BDC as it retained 87% of xylanase activity when incubated at 70 °C for 2 h as compared to 42% for XynZ-BDC. However, XynC-BC retained 70% activity on incubation at 70 °C for 2 h but XynC-C lost all activity under the same conditions. Km values for XynC-BC and XynC-C determined on soluble xylan were 3.1 and 3.6 mg ml−1, respectively, whereas these values for XynZ-BDC and XynZ-C were 33.3 and 15.4 mg ml−1, respectively. Thus the production of xylanase activity by expressing only the catalytic domains of XynC and XynZ is significantly enhanced.