Purification and characterization of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. G1

A cyclodextrin glucanotransferase (CGTase) was successively purified by ammonium sulphate precipitation, and affinity chromatography on a-CD (epoxy)-Sepharose 6B column. The specific activity of the CGTase was increased approximately 2200-fold, from 8.43 U/mg protein to 18,866 U/mg protein. SDS-PAGE showed that the purified CGTase was homogeneous and the molecular weight of the purified CGTase was about 75 kDa. The molecular weight of the enzyme that was estimated by gel filtration under native condition was 79 kDa. This has indicated that Bacillus sp. G1 CGTase is a monomeric protein. The isoelectric point (pI) of the enzyme was about 8.8. Characterization of the enzyme exhibited optimum pH and temperature of 6.0 and 60 8C, respectively. The enzyme was stable from pH 7.0 to 9.0 and retained its high activity up to 60 8C. However, in the presence of 20 mM Ca2+, the purified CGTase is able to prolong its thermal stability up to 70 8C. CGTase was strongly inhibited by ZnSO4, CuSO4, CoCl2, FeSO4, FeCl3 and EDTA. Km and Vmax for the purified enzyme were 0.15 mg/ml and 60.39 mg bcyclodextrin/( ml min), respectively, with soluble starch as substrate. In cyclodextrin production, tapioca starch was found to be the best substrate used to produce CDs. The enzyme produced g- and b-CD in the ratio of 0.11:0.89 after 24 h incubation at 60 8C, without the presence of any selective agents

Data for proteome analysis of Bacillus lehensis G1 in starch-containing medium

Bacillus lehensis G1 is a cyclodextrin glucanotransferase (CGTase) producer, which can degrade starch into cyclodextrin. Here, we present the proteomics data of B. lehensis 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 Escherichia coli” (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/)

Substrate and cofactor binding interaction studies of galactitol -1- Phosphate 5- Dehydrogenase from Peptoclostridium difficile

Tagatose is a high value low calorie sweetener that is used as a sugar substitute in the food and pharmaceutical industry. The production of tagatose requires the conversion of galactitol-1-phosphate to tagatose-6-phosphate by galactitol-1-phosphate 5-dehydrogenase (PdGPDH). Theobjective of this work is to study the protein-ligand interaction between PdGPDH and its ligands; galactitol-1-phosphate, Zn2+ and NAD+. Understanding of this mechanism will provide an insight into the possible catalytic events in these domains, thus providing information for potential protein engineering to improve the tagatose production. A 3D model of PdGPDH was constructed to identify the catalytic and coenzyme binding domains. In order to understand the interaction of PdGPDH with its ligands, a docking analysis of PdGPDH-substrate, PdGPDH-Zn2+ and PdGPDH-NAD+ complex was performed using CDOCKER in Discovery Studio 4.0 (DS 4.0). A series of docking events were performed to find the most stable binding interaction for the enzyme and its ligands. This study found that Cys 37, His 58, Glu 59, Glu 142 residues from PdGPDH form an active site pocket similar to known GPDH. A catalytic Zn2+ binding domain and a cofactor NAD+binding domain with strong hydrogen bonding contacts with the substrate and the cofactor were identified. The binding pockets of the enzyme for galactitol-1-phosphate, NAD+ and Zn2+has been defined. The stability of PdGPDH with its ligand was verified by utilizing the molecular dynamic simulation of docked complex. The results from this study will assist future mutagenesis study and enzyme modification work to improve the tagatose production

Cellobiohydrolase B of Aspergillus niger over-expressed in Pichia pastoris stimulates hydrolysis of oil palm empty fruit bunches

Background. Aspergillus niger, along with many other lignocellulolytic fungi, has been widely used as a commercial workhorse for cellulase production. A fungal cellulase system generally includes three major classes of enzymes i.e.,β-glucosidases, endoglucanases and cellobiohydrolases. Cellobiohydrolases (CBH) are vital to the degradation of crystalline cellulose present in lignocellulosic biomass. However, A. niger naturally secretes low levels of CBH. Hence, recombinant production of A. niger CBH is desirable to increase CBH production yield and also to allow biochemical characterisation of the recombinant CBH from A. niger. Methods. In this study, the gene encoding a cellobiohydrolase B (cbhB) from A. niger ATCC 10574 was cloned and expressed in the methylotrophic yeast Pichia pastoris X-33. The recombinant CBHB was purified and characterised to study its biochemical and kinetic characteristics. To evaluate the potential of CBHB in assisting biomass conversion, CBHB was supplemented into a commercial cellulase preparation (Cellic ® CTec2) and was used to hydrolyse oil palm empty fruit bunch (OPEFB), one of the most abundant lignocellulosic waste from the palm oil industry. To attain maximum saccharification, enzyme loadings were optimised by response surface methodology and the optimum point was validated experimentally. Hydrolysed OPEFB samples were analysed using attenuated total reflectance FTIR spectroscopy (ATR-FTIR) to screen for any compositional changes upon enzymatic treatment. Results. Recombinant CBHB was over-expressed as a hyperglycosylated protein attached to N-glycans. CBHB was enzymatically active towards soluble substrates such as 4-methylumbelliferylβ-D-cellobioside (MUC), p-nitrophenyl-cellobioside (pNPC) and p-nitrophenyl-cellobiotrioside (pNPG3) but was not active towards crystalline substrates like Avicel ® and Sigmacell cellulose. Characterisation of purified CBHB using MUC as the model substrate revealed that optimum catalysis occurred at 50 °C and pH 4 but the enzyme was stable between pH 3 to 10 and 30 to 80 °C. Although CBHB on its own was unable to digest crystalline substrates, supplementation of CBHB (0.37%) with Cellic ® CTec2 (30%) increased saccharification of OPEFB by 27%. Compositional analyses of the treated OPEFB samples revealed that CBHB supplementation reduced peak intensities of both crystalline cellulose Iα and Iβ in the treated OPEFB samples. Discussion. Since CBHB alone was inactive against crystalline cellulose these data suggested that it might work synergistically with other components of Cellic R CTec2. CBHB supplements were desirable as they further increased hydrolysis of OPEFB when the performance of Cellic® CTec2 was theoretically capped at an enzyme loading of 34% in this study. Hence, A. niger CBHB was identified as a potential supplementary enzyme for the enzymatic hydrolysis of OPEFB

Effect of the Cross Linkers’ Concentration on the Immobilization of Recombinant Escherichia Coli Cells on Hollow Fiber Membrane for Excretion of Cyclodextrin Glucanotransferase (CGTase)

The excretion of recombinant proteins into the culture medium is a preferred approach over cytoplasmic expression because of the high specific activity and ease of purification. However, cell lysis is one of a major problem during the excretion of recombinant protein due to the pressure build up in the periplasmic space through overproduction of the expressed recombinant protein. Cell immobilization is a promising solution for the improvement of protein excretion with reduction in cell lysis. In this study, the effect of cross linkers concentrations on the process of cell immobilization for the cyclodextringlucanotransferase (CGTase) excretion and cell viability were investigated. The hollow fiber membrane was treated using different concentrations (0.3, 0.67 and 1%) of cross linkers namely glutaraldehyde (GA), poly-L-lysine (PLL) and polyethylenimine (PEI). The optimum concentration of cross linkers was found to be 0.3% with the high excretion of CGTase. Interestingly, the immobilized cell on the non-treated hollow fiber membrane showed up to 15% increment of CGTase excretion with 55% reduction of cell lysis, as compared to the immobilized cells on the treated hollow fiber membrane. It could be concluded that, the low concentration of cross linkers exhibited the highest CGTase excretion. Moreover, the non-treated hollow fiber membrane is a promising approach for attachment of cells to the membrane without requiring treatment with any chemicals for the production of recombinant enzyme

Reaction optimization of Aspergillus niger α-L-arabinofuranosidase for improved arabinose production from kenaf stem

There are abundant of lignocellulosic biomass readily available with varying compositions. Kenaf (Hibiscus cannabinus) is one of this lignocellulosic biomass that has a high content of hemicellulose. This particular hemicellulose is composed of high arabinoxylan, which is a xylan backbone with arabinofuranosyl branches. In order to hydrolyze arabinoxylan, a branching enzyme is needed. Therefore, α-L-arabinofuranosidase from Aspergillus niger ATCC120120 (AnabfA) was used to hydrolyzed pre-treated kenaf and the reaction conditions were optimized using central composite design (CCD) to produce a significant amount of arabinose. There were 20 experiments conducted with 1.68 star points and 6 replicates at the centre points. The reaction conditions that were optimized are enzyme loading, substrate concentration and reaction time in which resulted with 88 U AnabfA activity, 0.9% (w/v) and 48 h, respectively. These optimized conditions managed to increase the yield of arabinose with 47.17 mg/g arabinose produced

Assessment of microwave-assisted pretreatments for enhancing pineapple waste delignification

Biological degradation of biomass for the production of fine chemicals is getting much interest nowadays. However, the complex and recalcitrance structure of the biomass hinders the success story of the degradation. Lignin is the main composition that impedes the bioconversion of biomass. Hence, an optimize delignification pretreatment need to be developed to enable the biological degradation process becomes much easier and thus higher production yield can be achieved afterwards. This study focuses on the assessment of pineapple waste (PW) delignification by applying microwave radiation on two different pretreated PW to facilitate the processes. The PW was initially pretreated with distilled water (dH2O) and peracetic acid (PAA) prior to the microwave radiation. Three main parameters (pretreatment time, min; temperature, °C; and microwave radiation power, W) were studied towards the effect of PW delignification. Lignin percentage before and after the pretreatments were compared and analysed. The results obtained revealed that the microwave-assisted PAA shows the best percentage of delignification compared to the microwave-assisted dH2O pretreatment. The best delignification process obtained in this study is a key indicator for a better biomass degradation to achieve higher yield of products in the future

Psychosocial issues of women with type 1 diabetes transitioning to motherhood: a structured literature review

BACKGROUND: Life transitions often involve complex decisions, challenges and changes that affect diabetes management. Transition to motherhood is a major life event accompanied by increased risk that the pregnancy will lead to or accelerate existing diabetes-related complications, as well as risk of adverse pregnancy outcomes, all of which inevitably increase anxiety. The frequency of hyperglycaemia and hypoglycaemia often increases during pregnancy, which causes concern for the health and physical well-being of the mother and unborn child. This review aimed to examine the experiences of women with T1DM focusing on the pregnancy and postnatal phases of their transition to motherhood. METHODS: The structured literature review comprised a comprehensive search strategy identifying primary studies published in English between 1990-2012. Standard literature databases were searched along with the contents of diabetes-specific journals. Reference lists of included studies were checked. Search terms included: 'diabetes', 'type 1', 'pregnancy', 'motherhood', 'transition', 'social support', 'quality of life' and 'psychological well-being'. RESULT: Of 112 abstracts returned, 62 articles were reviewed in full-text, and 16 met the inclusion criteria. There was a high level of diversity among these studies but three common key themes were identified. They related to physical (maternal and fetal) well-being, psychological well-being and social environment. The results were synthesized narratively. CONCLUSION: Women with type 1 diabetes experience a variety of psychosocial issues in their transition to motherhood: increased levels of anxiety, diabetes-related distress, guilt, a sense of disconnectedness from health professionals, and a focus on medicalisation of pregnancy rather than the positive transition to motherhood. A trusting relationship with health professionals, sharing experiences with other women with diabetes, active social support, shared decision and responsibilities for diabetes management assisted the women to make a positive transition. Health professionals can promote a positive transition to motherhood by proactively supporting women with T1DM in informed decision-making, by facilitating communication within the healthcare team and co-ordinating care for women with type 1 diabetes transitioning to motherhood

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

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