Abstract: Enzymes are the biocatalysts which catalyze the biochemical processes and thus have a wide variety of applications in the industrial sector. β-Galactosidase (E.C. 3.2.1.23) also known as lactase, is one of the prime enzymes, which has significant potential in the dairy and food processing industries. It has the capability to catalyze both the hydrolytic reaction for the production of lactose hydrolyzed milk and transgalactosylation reaction for the synthesis of prebiotics such as lactulose and galactooligosaccharides. These prebiotics have various nutritional and technological benefits. Although, the enzyme is naturally present in almonds, peaches, apricots and other variety of fruits and animals, the extraction of enzyme from these sources increases the cost of enzyme. Therefore, focus has been shifted towards the production of low cost enzyme from the microorganisms such as bacteria, yeast and fungi. As compared to yeast and bacteria, fungal β-galactosidase is generally preferred as being extracellular and thermostable in nature. Keeping the above in view, the present study was carried out for the isolation of the β-galactosidase producing fungal strain from the food as well as the agricultural wastes. A total of more than 100 fungal cultures were examined for their potential in enzyme production. All the fungal strains were screened using X-gal and IPTG as inducers in the modified Czapek Dox Agar medium. Among the various isolated fungal strains, the strain exhibiting the highest enzyme activity was chosen for further phenotypic and genotypic characterization. The strain was identified as Rhizomucor pusillus on the basis of 5.8s RNA gene sequencing data.
Abstract: Whey is the lactose rich by-product of the dairy
industry, having good amount of nutrient reservoir. Most abundant
nutrients are lactose, soluble proteins, lipids and mineral salts.
Disposing of whey by most of milk plants which do not have proper
pre-treatment system is the major issue. As a result of which, there
can be significant loss of potential food and energy source. Thus,
whey has been explored as the substrate for the synthesis of different
value added products such as enzymes. β-galactosidase is one of the
important enzymes and has become the major focus of research due
to its ability to catalyze both hydrolytic as well as
transgalactosylation reaction simultaneously. The enzyme is widely
used in dairy industry as it catalyzes the transformation of lactose to
glucose and galactose, making it suitable for the lactose intolerant
people. The enzyme is intracellular in both bacteria and yeast,
whereas for molds, it has an extracellular location. The present work
was carried to utilize the whey for the production of β-galactosidase
enzyme using both yeast and fungal cultures. The yeast isolate
Kluyveromyces marxianus WIG2 and various fungal strains have
been used in the present study. Different disruption techniques have
also been investigated for the extraction of the enzyme produced
intracellularly from yeast cells. Among the different methods tested
for the disruption of yeast cells, SDS-chloroform showed the
maximum β-galactosidase activity. In case of the tested fungal
cultures, Aureobasidium pullulans NCIM 1050 was observed to be
the maximum extracellular enzyme producer.
Abstract: The hydrolysis of lactose using β-galactosidase is one of the most promising biotechnological applications, which has wide range of potential applications in food processing industries. However, due to intracellular location of the yeast enzyme, and expensive extraction methods, the industrial applications of enzymatic hydrolysis processes are being hampered. The use of permeabilization technique can help to overcome the problems associated with enzyme extraction and purification of yeast cells and to develop the economically viable process for the utilization of whole cell biocatalysts in food industries. In the present investigation, standardization of permeabilization process of novel yeast isolate was carried out using a statistical model approach known as Response Surface Methodology (RSM) to achieve maximal b-galactosidase activity. The optimum operating conditions for permeabilization process for optimal β-galactosidase activity obtained by RSM were 1:1 ratio of toluene (25%, v/v) and ethanol (50%, v/v), 25.0 oC temperature and treatment time of 12 min, which displayed enzyme activity of 1.71 IU /mg DW.
Abstract: Bones are dynamic and responsive organs, they
regulate their strength and mass according to the loads which they are subjected. Because, the Wnt/β-catenin pathway has profound
effects on the regulation of bone mass, we hypothesized that mechanical loading of bone cells stimulates Wnt/β-catenin signaling, which results in the generation of new bone mass.
Mechanical loading triggers the secretion of the Wnt molecule, which after binding to transmembrane proteins, causes GSK-3β (Glycogen synthase kinase 3 beta) to cease the phosphorylation of β-catenin. β-catenin accumulation in the cytoplasm, followed by its
transport into the nucleus, binding to transcription factors (TCF/LEF)
that initiate transcription of genes related to bone formation. To test this hypothesis, we used TOPGAL (Tcf Optimal Promoter
β-galactosidase) mice in an experiment in which cyclic loads were
applied to the forearm. TOPGAL mice are reporters for cells effected
by the Wnt/β-catenin signaling pathway. TOPGAL mice are genetically engineered mice in which transcriptional activation of β-
catenin, results in the production of an enzyme, β-galactosidase. The
presence of this enzyme allows us to localize transcriptional
activation of β-catenin to individual cells, thereby, allowing us to quantify the effects that mechanical loading has on the Wnt/β-catenin pathway and new bone formation. The ulnae of loaded TOPGAL
mice were excised and transverse slices along different parts of the
ulnar shaft were assayed for the presence of β-galactosidase.
Our results indicate that loading increases β-catenin transcriptional
activity in regions where this pathway is already primed (i.e. where basal activity is already higher) in a load magnitude dependent
manner. Further experiments are needed to determine the temporal and spatial activation of this signaling in relation to bone formation.