Imdad Kaleem

Degumming of vegetable oils by a novel phospholipase B from Pseudomonas fluorescens BIT-18.

Pseudomonas fluorescens BIT-18 was isolated from soil near a vegetable oil factory and shown to produce a B-type phospholipase. The enzyme was partially purified by ammonium sulfate precipitation. Gas chromatography demonstrated that the enzyme preparation hydrolyzed both the 1- and 2-ester bonds of phosphatidylcholine. When degumming of soybean, rapeseed, and peanut oil was performed with this enzyme preparation, oils with phosphorous contents lower than 5mg/kg were obtained after 5h of enzyme treatment at 40°C. The enzyme preparation did not show lipase activity, thus free fatty acids were only generated from the phospholipids. Therefore, this novel phospholipase B is potentially useful for the refining of high-quality oils with attractive yields.

N-glycosylation enhances functional and structural stability of recombinant β-glucuronidase expressed in Pichia pastoris

Recombinant β-glucuronidase (GUS) expressed in Pichia pastoris GS115 is an important glycoprotein, encoded by a gene with four potential N-glycosylation sites. To investigate the impact of N-linked carbohydrate moieties on the stability of recombinant GUS, it was deglycosylated by peptide-N-glycosidase F (PNGase-F) under native conditions. The enzymatic activities of the glycosylated and deglycosylated GUS were compared under various conditions such as temperature, pH, organic solvents, detergents and chaotropic agent. The results demonstrated that the glycosylated GUS retained greater fraction of maximum enzymatic activity against various types of denaturants compared with the deglycosylated. The conformational stabilities of both GUS were analyzed by monitoring the unfolding equilibrium by using the denaturant guanidinium chloride (dn-HCl). The glycosylated GUS displayed a significant increase in its conformational stability than the deglycosylated counterpart. These results affirmed the key role of N-glycosylation on the structural and functional stability of β-glucuronidase and could have potential applications in the functional enhancement of industrial enzymes.

N-linked glycosylation influences on the catalytic and biochemical properties of Penicillium purpurogenum β-d-glucuronidase

To study the influence of N-linked carbohydrate moiety on the catalytic and biochemical properties of glycosylated enzyme, a recombinant β-d-glucuronidase (PGUS-P) from Penicillium purpurogenum as a model glycoprotein, was deglycosylated with peptide-N-glycosidase F (PNGase-F) under native conditions. The enzymatic deglycosylation procedure resulted in the complete removal of carbohydrate moiety. Compared with the glycosylated PGUS-P, the deglycosylated PGUS-P exhibited 20-70% higher activity (p<0.05) within pH 6-9, but 15-45% lower activity (p<0.05) at 45-70°C. The apparent decrease in the thermal stability of the deglycosylated enzyme was reflected by a decrease in the denaturation temperature (T(d)) values determined by differential scanning calorimetry (DSC). The removal of N-linked glycans also reduced enzymes sensitivity to certain metal ions. The deglycosylated PGUS-P displayed lower K(m) vaules, but higher k(cat)/K(m) ratios than the glycosylated isoform towards glycyrrhizin. The consequent conformational changes were also determined by circular dichroism (CD) and fluorescence spectroscopy which revealed no significant difference in the secondary but a slight dissimilarity between the tertiary structures of both isoforms of PGUS-P.

Transcriptional Profiling of Protein Expression Related Genes of Pichia pastoris under Simulated Microgravity

The physiological responses and transcription profiling of Pichia pastoris GS115 to simulated microgravity (SMG) were substantially changed compared with normal gravity (NG) control. We previously reported that the recombinant P. pastoris grew faster under SMG than NG during methanol induction phase and the efficiencies of recombinant enzyme production and secretion were enhanced under SMG, which was considered as the consequence of changed transcriptional levels of some key genes. In this work, transcriptiome profiling of P. pastoris cultured under SMG and NG conditions at exponential and stationary phases were determined using next-generation sequencing (NGS) technologies. Four categories of 141 genes function as methanol utilization, protein chaperone, RNA polymerase and protein transportation or secretion classified according to Gene Ontology (GO) were chosen to be analyzed on the basis of NGS results. And 80 significantly changed genes were weighted and estimated by Cluster 3.0. It was found that most genes of methanol metabolism (85% of 20 genes) and protein transportation or secretion (82.2% of 45 genes) were significantly up-regulated under SMG. Furthermore the quantity and fold change of up-regulated genes in exponential phase of each category were higher than those of stationary phase. The results indicate that the up-regulated genes of methanol metabolism and protein transportation or secretion mainly contribute to enhanced production and secretion of the recombinant protein under SMG.

Effects of a Non-Conservative Sequence on the Properties of β-glucuronidase from Aspergillus terreus Li-20

We cloned the β-glucuronidase gene (AtGUS) from Aspergillus terreus Li-20 encoding 657 amino acids (aa), which can transform glycyrrhizin into glycyrrhetinic acid monoglucuronide (GAMG) and glycyrrhetinic acid (GA). Based on sequence alignment, the C-terminal non-conservative sequence showed low identity with those of other species; thus, the partial sequence AtGUS(-3t) (1–592 aa) was amplified to determine the effects of the non-conservative sequence on the enzymatic properties. AtGUS and AtGUS(-3t) were expressed in E. coli BL21, producing AtGUS-E and AtGUS(-3t)-E, respectively. At the similar optimum temperature (55°C) and pH (AtGUS-E, 6.6; AtGUS(-3t)-E, 7.0) conditions, the thermal stability of AtGUS(-3t)-E was enhanced at 65°C, and the metal ions Co2+, Ca2+ and Ni2+ showed opposite effects on AtGUS-E and AtGUS(-3t)-E, respectively. Furthermore, Km of AtGUS(-3t)-E (1.95 mM) was just nearly one-seventh that of AtGUS-E (12.9 mM), whereas the catalytic efficiency of AtGUS(-3t)-E was 3.2 fold higher than that of AtGUS-E (7.16 vs. 2.24 mM s−1), revealing that the truncation of non-conservative sequence can significantly improve the catalytic efficiency of AtGUS. Conformational analysis illustrated significant difference in the secondary structure between AtGUS-E and AtGUS(-3t)-E by circular dichroism (CD). The results showed that the truncation of the non-conservative sequence could preferably alter and influence the stability and catalytic efficiency of enzyme.

Preparative Enrichment and Separation of Glycyrrhetinic Acid Monoglucuronide from Fermentation Broths with Macroporous Resins

Preparative enrichment and separation of Glycyrrhetinic acid monoglucuronide (GAMG) from the pretreated fermentation broth of glycyrrhizin was studied by using six macroporous resins with different physical and chemical properties. D101 resin showed the maximum effectiveness among the tested resins. The solute affinity towards D101 resin at different temperatures was described in terms of Langmuir and Freundlich isotherms, and the equilibrium experimental data were well-fitted to the two isotherms. The dynamic adsorption and desorption tests were carried out in order to optimize the operational parameters for the efficient separation of GAMG. After one run treatment with D101 resin, the contents of GAMG in the product were increased to 8.2-fold with recovery yields of 93.3%. The process achieved easy and effective enrichment and separation of GAMG with D101 resin, and it could be applied for the large-scale preparation of GAMG from the fermentation broth of glycyrrhizin.

Application Efficacy of Biological Seed Coating Agent from Combination of PGPR on Cotton in the Field

Application efficacy of the biological seed coating agent (BSCA) prepared with polyvinyl alcohol, sodium carboxymethyl celullose and Na-bentonite and having plant growth-promoting rhizobacteria (PGPR) Rs-5 and BCL-8, was investigated on cotton plants in the field. The results showed that the germination rate of cotton seeds treated with BSCA was increased by 11.3%, but the incidence rate of cotton seedlings was decreased by 10.8%, while other growth factors such as plant height, fresh weight, dry weight and leaf area of cotton seedlings were increased by 14.4%, 19.1%, 25.7% and 47.4% compared to treated with chemical seed coating agent (CSCA), respectively. Further analysis demonstrated that enzymatic activity of PAL, POD, PPO, SOD and photosynthetic pigment contents were improved to a certain extent as well, while MDA activity was declined. Moreover, the ionic uptake of Ca2+, Mg2+ and K+ in cotton was enhanced, while Na+ absorption was reduced in BSCA compared to CSCA. The yield of cotton seeds treated with BSCA was increased up to 10.5% in comparison with CSCA treatment. Hence, our results revealed that the use of BSCA could not only alleviate the adverse effects of salinity but also has a promising plant growth promoting potential in salinized soil.

Antibacterial Activities of Chemical and Biological Food Preservatives towards Food Spoilage Bacillus among and within Species

Food preservatives could cause a wide spectrum of antibacterial efficacies against different spoilage microbial species or strains; however, the mode of variation in antibacterial activities among and within species is still unclear which limits their effective applications in food preservation. In this study, ten thermoresistant spore-forming Bacillus strains as a typical spoilage microbial group were used to evaluate the variances of antibacterial activities of three chemical and three biological food preservatives among these species or strains by the statistical analysis of MIC using SAS 9.2 software. The biological preservatives exhibited much higher anti-bacillus activities compared to the chemical preservatives, and significant variances of MICs were observed among different Bacillus species for chemical preservatives while no variances were observed for biological preservatives. Variances of MICs among different strains in a species were not observed for both chemical and biological preservatives. The possible antibacterial mechanism has also been discussed to encounter food spoilage.