Different Approaches For Protein Engineering In Industrial Biotechnology

Protein engineering is the novel field which has wide applications from pharmaceutics, industry, commercial, laundry and research. It may apply rational design or non rational design or both. Site directed mutagenesis is a classical approach involving the protein folding principles and as such different techniques involving multidisciplinary research and broad knowledge is required involving biocomputing of complex data obtained from various sequencing projects and prediction of the future protein structure either chemically or genetically modified. Non rational mutagenesis or directed evolution involves random mutations in the gene encoding protein or shuffling the genes encoding different domains producing a random set of numerous large libraries of mutant proteins, using advanced technology the desired protein can be selected but the exact structure or changes may remain unnoticed

Molecular Bio-imprinting of Biocatalysts

Energy conservation is the cry of the day. Attempts are made all over the world to occupy and use energy reserves. Increased industrialization and mechanization has led to the depletion of natural energy reserves. Its unavoidable to search for renewable sources of energy, which may be not used now but can be used by future generations. We are using the expertise of our ancestors. Thus exploiting the nature and newer techniques in this area would yield the best results. Bio-imprinting is one of those techniques whereby chemical modification is done in order to achieve highly expressed protein which can be stored in its highly active form in the specific solvent

Mass transfer efficiency of a tall and low plate free area liquid pulsed sieve-plate extraction column

Acknowledgements The authors would like to acknowledge Chakwal group of industries for funding the project. Ms. Madiha, Ms. Zona, Mr. Sohaib, Mr. Abdullah, Mr. Mudassar, and Mr. Salahuddin also deserve our acknowledgements for their assistance in different ways.Peer reviewedPublisher PD

Expression and sequence characterization of growth hormone binding protein of Nili-Ravi buffaloes (Bubalus bubalis)

The growth hormone binding protein (GHBP) was isolated from the liver of Nili-Ravi buffaloes (Bubalus bubalis), reverse transcriptase-polymerase chain reaction (RT-PCR) amplified and sequence characterized. RT-PCR analysis demonstrated high degree sequence identities (97.3 to 99.6%) of BbGHBP cDNA with Bos taurus, Ovis aries and Capra hircus. An expression plasmid was constructed for the production of BbGHBP in Escherichia coli BL21 (RIPL) CodonPlus under the control of T7lac promoter. On induction with isopropyl β-D thiogalactopyranoside, the BbGHBP was expressed at levels >30% of the total E. coli proteins. The target protein expressed as inclusion bodies was solubilized in denaturing solution and refolded by step/pulsatile dilution method using cysteine and cystine redox potential. Purification to near homogeniety (>98%) was achieved by ion-exchange chromatography with a recovery yield of 64%. Mass spectrometric analysis of the purified BbGHBP showed a single peak of 30,756 Da. A radioprotein assay evaluated the binding affinity of recombinant BbGHBP with iodinated bovine growth hormone (bGH) which demonstrated active conformation of BbGHBP. These results demonstrate high expression and sequence characterization of BbGHBP in Nili-Ravi buffaloes and provide the basis for the assessment of BbGHBP in other breeds of buffalo.Keywords: Liver, Nili-Ravi buffalo, GHBP, MALDI-TOF mass spectrometry, radioprotein binding assay, refoldin

Surgical Outcome of Endoscopic Third Ventriculostomy in Patients Having High ETV Success Score: One-Year Experience at a Tertiary Care Hospital

Background & Objective:  Endoscopic third Ventriculostomy (ETV) is an accepted alternative to VP shunt in patients with obstructive hydrocephalus. We will share our experience and outcome. Materials & Methods:  Thirty consecutive ETV cases performed by a single surgeon during 1 year in patients with an ETV success score of 60 or higher were included in this study. Patients’ demographics, outcomes, and complications are reported. Results:  (60%) were male and 12 (40%) were female. The mean age in our study was 6.1 years ± 9 (mean ± SD). Posterior fossa tumor was the most common etiology in our series (46.6%) followed by aqueductal stenosis (23.3%). Eighty percent of our patients did not experience an ETV failure. The complication rate was 20%. Inadequate ventriculostomy in 6.6% of the patients was the commonest complication. Conclusion:  ETV is safe and effective in patients with high ETV success scores

Firm Size as Moderator to Leverage-Performance Relation: An Emerging Market Review

Present study explored leverage-performance relation while the moderating firm size in developing countries like Pakistan. Data is collected for 304 Pakistani non-financial firms for the period of 2005-2013. It is found that overall leverage-performance relation is negative for all types of firms. However, such losses are more prominent for small size firms. Results also showed that the leverage-performance relation is nonlinear for medium and large size firms.However, in practice these firms are not targeting optimal level and over-leveraging that ultimately decrease their profits.So, financial managers of small size firms should avoid debt financing while for large and medium size firms,managers need to adjust their debt ratio toits optimal level. Keywords: Leverage, Performance, Capital Structure, Moderation, Firm Size JEL code: G32

Proizvodnja i karakterizacija α-galaktozidaze fermentacijom na čvrstoj podlozi s pomoću višestruko mutiranog soja Aspergillus niger

α-Galactosidase is applied in the sugar industry to enhance sugar recovery from sugar beet syrup and to improve nutritional value of the soymilk. In the present investigation, the influence of process variables on the production of this important enzyme has been explored in a newly isolated multiple mutant strain of Aspergillus niger in solid-state fermentation (SSF). Defined fermentation parameters include substrate type (pure lactose and by-products of rice and flour mills as prime substrates), nitrogen source, incubation time, initial pH of the medium and incubation temperature. Extracellular α-galactosidase reached the value of 135.4 IU/g of dry substrate (IU/g) after 96 h of fermentation. Supplementation with 2 g of glucose and 3 g of corn steep liquor significantly increased the enzyme production, and maximum value of product yield (318 IU/g) by the mutant strain was significantly higher than that reported by the wild type (this work), or other A. niger mutants, recombinants and yeasts reported in literature as producers of elevated levels of α-galactosidase. Among three α-galactosidases, one possessing high subunit molecular mass proteins (99 and 100 kDa) has been characterized in both wild and mutant organisms. Thermal properties of the purified enzymes indicate that the mutation decreased the values of activation energy for the formation of enzyme-substrate (ES) complex, enthalpy, Gibbs free energy demand for substrate binding, and transition state stabilization. A thermodynamic study of irreversible inactivation of enzymes suggests that the mutant–derived enzyme is more thermostable than the native enzyme, which is attributable to amino acids involved in active catalysis. Because of these properties, the mutant organism is a novel organism and may be exploited for bulk production of thermostable α-galactosidase for the above industrial and nutritional applications.U industriji se α-galaktozidaza primjenjuje radi povećanja prinosa šećera iz sirupa šećerne repe te poboljšanja hranjive vrijednosti sojinog mlijeka. U radu je ispitan utjecaj varijabli procesa na proizvodnju ovog važnog enzima fermentacijom na čvrstoj podlozi s pomoću novoizoliranog višestruko mutiranog soja Aspergillus niger. Procijenjeni su sljedeći parametri: supstrat (čista laktoza te nusprodukti meljave riže i brašna), izvor dušika, vrijeme inkubacije, početna pH-vrijednost podloge i temperatura inkubacije. Nakon 96 sati fermentacije dobiveno je 135,4 IU ekstracelularne galaktozidaze po g suhe podloge. Dodatak od 2 g glukoze i 3 g kukuruznog ekstrakta značajno je povećao proizvodnju enzima. S pomoću mutantnog soja postignut je kudikamo veći maksimalni prinos (318 IU/g) nego s divljim sojem (u ovom radu) ili drugim u literaturi navedenim mutantnim sojevima A. niger, rekombinantnim vrstama ili kvascima koji proizvode α-galaktozidazu. Ispitane su tri α-galaktozidaze, od kojih je ona što sadrži podjedinice proteina velike molekularne mase (99 i 100 kDa) karakterizirana i u divljem i u mutantnom soju. Toplinska svojstva pročišćenih enzima pokazuju da je mutacija smanjila energiju aktivacije potrebnu za nastajanje kompleksa enzim-supstrat, entalpiju, količinu Gibbsove slobodne energije utrošene za vezivanje supstrata i stabilizaciju prijelaznog stanja. Termodinamičkim ispitivanjem ireverzibilne inaktivacije enzima zaključeno je da enzim izoliran iz mutantnog soja ima veću termostabilnost od prirodnog enzima zbog aminokiselina u aktivnom katalitičkom procesu. Zbog toga bi se svojstva mutantni organizam mogao upotrijebiti u proizvodnji veće količine termostabilne α-galaktozidaze, za njezinu primjenu u industriji šećera, te radi poboljšanja hranjive vrijednosti proizvoda

Carrot and Stick Approach: The Exploitative Leadership and Absenteeism in Education Sector

Utilizing the conservation of resources theory, this study investigates serial mediation of facades of conformity and depression between exploitative leadership and absenteeism. A total of 211 education sector employees using the convenient sampling technique took part in the survey with data collected in a time-lagged research design. Findings of the study reveal that facades of conformity and depression mediate the independent paths and play a serial mediating role between EL and absenteeism path. This study suggests that EL works as a workplace stressor, under which employees try to protect their valuable resources from further loss in the form of facades of conformity, in doing so, it leads to depression; thus, employees ultimately use absenteeism as an active coping strategy to cope with workplace stressors

Proizvodnja i karakterizacija α-galaktozidaze fermentacijom na čvrstoj podlozi s pomoću višestruko mutiranog soja Aspergillus niger

α-Galactosidase is applied in the sugar industry to enhance sugar recovery from sugar beet syrup and to improve nutritional value of the soymilk. In the present investigation, the influence of process variables on the production of this important enzyme has been explored in a newly isolated multiple mutant strain of Aspergillus niger in solid-state fermentation (SSF). Defined fermentation parameters include substrate type (pure lactose and by-products of rice and flour mills as prime substrates), nitrogen source, incubation time, initial pH of the medium and incubation temperature. Extracellular α-galactosidase reached the value of 135.4 IU/g of dry substrate (IU/g) after 96 h of fermentation. Supplementation with 2 g of glucose and 3 g of corn steep liquor significantly increased the enzyme production, and maximum value of product yield (318 IU/g) by the mutant strain was significantly higher than that reported by the wild type (this work), or other A. niger mutants, recombinants and yeasts reported in literature as producers of elevated levels of α-galactosidase. Among three α-galactosidases, one possessing high subunit molecular mass proteins (99 and 100 kDa) has been characterized in both wild and mutant organisms. Thermal properties of the purified enzymes indicate that the mutation decreased the values of activation energy for the formation of enzyme-substrate (ES) complex, enthalpy, Gibbs free energy demand for substrate binding, and transition state stabilization. A thermodynamic study of irreversible inactivation of enzymes suggests that the mutant–derived enzyme is more thermostable than the native enzyme, which is attributable to amino acids involved in active catalysis. Because of these properties, the mutant organism is a novel organism and may be exploited for bulk production of thermostable α-galactosidase for the above industrial and nutritional applications.U industriji se α-galaktozidaza primjenjuje radi povećanja prinosa šećera iz sirupa šećerne repe te poboljšanja hranjive vrijednosti sojinog mlijeka. U radu je ispitan utjecaj varijabli procesa na proizvodnju ovog važnog enzima fermentacijom na čvrstoj podlozi s pomoću novoizoliranog višestruko mutiranog soja Aspergillus niger. Procijenjeni su sljedeći parametri: supstrat (čista laktoza te nusprodukti meljave riže i brašna), izvor dušika, vrijeme inkubacije, početna pH-vrijednost podloge i temperatura inkubacije. Nakon 96 sati fermentacije dobiveno je 135,4 IU ekstracelularne galaktozidaze po g suhe podloge. Dodatak od 2 g glukoze i 3 g kukuruznog ekstrakta značajno je povećao proizvodnju enzima. S pomoću mutantnog soja postignut je kudikamo veći maksimalni prinos (318 IU/g) nego s divljim sojem (u ovom radu) ili drugim u literaturi navedenim mutantnim sojevima A. niger, rekombinantnim vrstama ili kvascima koji proizvode α-galaktozidazu. Ispitane su tri α-galaktozidaze, od kojih je ona što sadrži podjedinice proteina velike molekularne mase (99 i 100 kDa) karakterizirana i u divljem i u mutantnom soju. Toplinska svojstva pročišćenih enzima pokazuju da je mutacija smanjila energiju aktivacije potrebnu za nastajanje kompleksa enzim-supstrat, entalpiju, količinu Gibbsove slobodne energije utrošene za vezivanje supstrata i stabilizaciju prijelaznog stanja. Termodinamičkim ispitivanjem ireverzibilne inaktivacije enzima zaključeno je da enzim izoliran iz mutantnog soja ima veću termostabilnost od prirodnog enzima zbog aminokiselina u aktivnom katalitičkom procesu. Zbog toga bi se svojstva mutantni organizam mogao upotrijebiti u proizvodnji veće količine termostabilne α-galaktozidaze, za njezinu primjenu u industriji šećera, te radi poboljšanja hranjive vrijednosti proizvoda

Protein Engineering of Endoglucanase CelR of Clostridium thermocellum for Enhanced Expression

Background: Enhanced production and improved properties of cellulases for a greater activity on plant biomass would rank amongst the top priorities for second-generation ethanol production. Based on the emergence of protein engineering as a cutting-edge technology for enhancing enzyme activity and expression level, the present study is aimed at the application of this technique to the major cellulosomal processing endoglucanase of C. thermocellum, CelR for refining enzyme characteristics. Methods: The full-length native enzyme gene (CelR) and a truncated version without the docking domains at C-terminus (CelR-CB) were PCR amplified using gene specific primers. The amplified PCR products were T/A cloned in the vector pTZ57 R/T and transformed in E. coli DH5α. The cellulase genes from the confirmed transformed plasmids were sub-cloned in T7 promoter-based expression vector pET-28a and expression analysis was done in E. coli (DE3) BL21 codon Plus. Results: An SDS PAGE analysis of both the CelR derivatives revealed that the truncated version i.e. CelR-CB showed a two-fold increase in expression level as compared to the full-length enzyme. Conclusion: The increased expression level of CelR in E. coli coupled with its increased production therefore makes it a promising method for augmenting the recombinant enzyme production for potential applications.