Whole genome shot gun sequence analysis of L. fermentum MTCC 8711 (GenBank Acc. No. AP008937) showed the presence of two bsh genes designated as bsh1 and bsh2 respectively. The 927 nucleotide long bsh1 gene, though exhibited 99% similarity with L. fermentum F1 bile salt hydrolase (bsh) gene with the Nucleotide BLAST analysis, did not produce any promising activity with cloning and expression experimenting. Bsh2 was 978 bp long and showed 99% homology with the bsh gene of L. fermentum NCDO394. Primers were designed to amplify the bsh2 gene from the genome of L. fermentum MTCC 8711 with the NcoI and AscI restriction fragments introduced in them. PCR amplification with these set of primers resulted in 1kb amplicon.
Over expression of BSH in native L. fermentum MTCC 8711
Recombinant L. fermentum MTCC 8711 harbouring pBSHCM were studied for over expression of the bile salt hydrolase gene. Expression was induced with 0.5, 1, 2 and 3 percentages of xylose for 4h. Extracellular and intracellular fractions were collected and quantitated using ninhydrin assay and SDS-PAGE analysis. For extracellular expression of bile salt hydrolase 2% of xylose was found to be optimal concentration, resulting with 0.5 enzyme activity (U/ml) as shown in Figure 1. In the case of intracellular expression, 0.23 U/ml of maximal enzyme activity was obtained with 1% xylose concentration. Extra cellular bile salt hydrolase enzyme activity was found to be predominant over the intracellular expression. Furthermore enzyme activity was found to be more specific towards the glyco conjugated bile salt upon the tauro conjugates (Data not shown). Extra cellular fractions with different concentrations of xylose induction were resolved on SDS-PAGE. The coomassie stained...
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...he functionality of the bsh genes of Lactobacillus plantarum WCFS1 was explored using Bsh-deficient host Lactococcus. lactis NZ9000 by use of the NICE system (Lambert et al 2008). Few reports are also available on the heterologous expression of the probiotic lactobacillus strains in other host strains other than Lactobacillus such as E. coli (Oh et al 2008, Kumar et al 2013). However no reports are found on the overexpression of the bsh gene in the native host Lactobacillus spp. Since Lactobacilli are very much employed in the fermented food industry, preparation of fermented foods such as yoghurt using bsh active probiotic strains would serve as a finctional biotherapeutic. Therefore, use of recombinant bsh overproducing Lactobacillus fermentum MTCC 8711 could be a potential probiotic strain that could be applies as a biotherapeutic to overcome hypercholesteremia.
The isolate possesses some enzymes required for hydrolytic reactions. Hydrolytic enzymes found to be secreted from the bacterium, are -amylase, casein, and PYRase. In the starch hydrolysis and casein tests, there was a zone of clearing around the bacterium, which was indicative of the secreted enzymes necessary to break down starch and casein. In the PYR test, the presence of PYRase was detected by a color change to red on the PYR disc after the addition of the PYR reagent (p-dimethylaminocinnamaldehyde). Hydrolytic enzymes for which the EI tested negative were urease, gelatinase, and DNAse. In the Urea Hydrolysis test, it was observed that the urea broth did not have a color change, indicating that there was no urease secreted to break down urea in the broth. Similarly, there was no gelatinase present to break down gelatin in the Gelatin Hydrolysis test, so the nutrient gelatin remained solid. It was concluded that the EI does not possess DNase because there was no clearing zone around the bacteria, indicating that DNA had not been
Living organisms undergo chemical reactions with the help of unique proteins known as enzymes. Enzymes significantly assist in these processes by accelerating the rate of reaction in order to maintain life in the organism. Without enzymes, an organism would not be able to survive as long, because its chemical reactions would be too slow to prolong life. The properties and functions of enzymes during chemical reactions can help analyze the activity of the specific enzyme catalase, which can be found in bovine liver and yeast. Our hypothesis regarding enzyme activity is that the aspects of biology and environmental factors contribute to the different enzyme activities between bovine liver and yeast.
Streak plate technique was used to isolate pure culture for each bacteria (2). The Gram stain was used to determine Gram reaction and morphology of each bacteria (2) Selective and differential media such as, salt agar, MacConkey agar and blood agar were used for bacterial identification (2). Gelatin deeps were inoculated to detect production of gelatinase (2). Starch Agar plate were inoculated to detect amylase (2). Ocular reticle used to determine bacteria size (2). Motility deeps were inoculated to detect motility on bacteria (2). Thioglycollate broth used to determine oxygen requirements (2). Carbohydrate fermentation
The aim of this experiment was to isolate cDNA molecule CIH-1 (Colletotrichum lindemuthianum CIH1 gene) that is contained in vector pBK-CMV and transfer it into cloning vector pUC19. This was attempted by conducting a restriction digest of vectors pUC19 and pBk-CMV containing CIH-1, using restriction endonucleases Xba1 and EcoR1 and the characterization of recombinant plasmids.
Glase, Jon C. A Study of Gene Linkage and Mapping Using Tetrad Analysis in the Fungus
The expression of lac operon in each tube equals the amount of beta-galactosidase produced. Therefore, by looking at the amount of beta-galactosidase under different conditions collectively is a good way to understand the function of inducers and repressors in supervising the expression of lac operon and the control of gene expression generally.
The purpose of this experiment was to discover the specificity of the enzyme lactase to a spec...
An Investigation into the Effect of Lipase Concentration on the Hydrolysis Of Fats Using the data loggers a recording of the pH was taken every 5 seconds and for each experiment the data loggers produced graphs of the change in pH. From each of these graphs a gradient was calculated which showed the rate of pH change per second. Firstly I calculated the gradients by choosing the steepest section of the graph and dividing the change in pH of this section by the time. However this method proved to be quite inaccurate giving very varied results, for example in these results the average rate of reaction for the 4% lipase solution (-0.457 pH/min) was lower than the 3% lipase solution (-0.471 pH/min). Also the rates in the 2% lipase solution ranged from -0.01 pH/min to -0.95 pH/min showing little reliability in the results. This was partly as I was only guessing which the steepest part of the graph was.
The independent variable for this experiment is the enzyme concentration, and the range chosen is from 1% to 5% with the measurements of 1, 2, 4, and 5%. The dependant variable to be measured is the absorbance of the absorbance of the solution within a colorimeter, Equipments: Iodine solution: used to test for present of starch - Amylase solution - 1% starch solution - 1 pipette - 3 syringes - 8 test tubes – Stop clock - Water bath at 37oc - Distilled water- colorimeter Method: = == ==
Enteric bacteria are major microorganisms that colonise human’s gastrointestinal tract- a long tube containing specialised sections such as the mouth, oesophagus, stomach, intestines, rectum and the anus. Gut bacteria make up approximately 95 percent of the total cells in the body, with the colon containing the densest microbial communities (Gibson, Rastall and Fuller 2008: 52). Human gastrointestinal tract consists of 100 different prokaryotic species, and with two bacterial species, firmicutes and bacteroicidetes dominating them (Brooker et al. 2011: 559).
In this experiment as a whole, there were three individual experiments conducted, each with an individualized hypothesis. For the effect of temperature on enzyme activity, catalase activity will be decreased when catalase is exposed to temperatures greater than or less approximately 23 degrees Celsius. For the effect of enzyme concentration on enzyme activity, a concentration of greater or less than approximately 50% enzymes, the less active catalase will be. Lastly, the more the pH buffer deviates from a basic pH of 7, the less active catalase will be.
Abstract: Enzymes are catalysts therefore we can state that they work to start a reaction or speed it up. The chemical transformed due to the enzyme (catalase) is known as the substrate. In this lab the chemical used was hydrogen peroxide because it can be broken down by catalase. The substrate in this lab would be hydrogen peroxide and the enzymes used will be catalase which is found in both potatoes and liver. This substrate will fill the active sites on the enzyme and the reaction will vary based on the concentration of both and the different factors in the experiment. Students placed either liver or potatoes in test tubes with the substrate and observed them at different temperatures as well as with different concentrations of the substrate. Upon reviewing observations, it can be concluded that liver contains the greater amount of catalase as its rates of reaction were greater than that of the potato.
Lactic acid have more growth requirements than then normal bacteria since it was evolved in nutrient-rich environments. Lactic acid bacteria have diverse mechanisms for creating the energy needed to support and sustain biological activities. The availability of organic acid in the fruit can be important in allowing growth and metabolism. As lactic acid bacteria have the ability to produce large amount of acids, they often inhibit the development of other bacteria in juices and are able to cause their own autolysis. Excessive clarification and pre treatment of the fruit during the process of sending the fruit to the market which removes many of the natural yeasts and flora. The chemical compsition of juice also affect the rate of fermentation. Fruits generally tend to contain sufficient substrate (soluble sugars)that allow for the yeast and bacteria to fermented , so it can be said that because the fruits used did not show a very high increase in acidity it did not provide a sufficient substrate for the lactic acid bacteria that is present on the fruit to be used for fermentation.Temperature has an impact on the growth and activity of different strains of yeast. At temperatures of
To fulfill the increasing worldwide demand and to reduce the cost of vitamins different group of researchers are using different microorganisms for the production of vitamins like vitamin B12, vitamin C, vitamin B2 and vitamin D. Here in this review I have mentioned some of them which are producing good yield of vitamins.
Microbes are major key components in both are homes and industrial food preparation. There are number of lactic acid which is a form of bacteria which is a large group of beneficial bacteria used in certain foods while they are getting prepared such as yogurt, cheese, sour cream, butter milk and other type of fermented milk products. Things such as vinegars are produced by bacterial acetic acid fermentation. Yeast is also major use in the making of beer and wine and also for the leaving of breads. This also involves fermentations to convert corn and other vegetable carbohydrates to also make beer, wine or gasohol but also bacteria is the agents of are other foods. Other fermented foods will include things such as soy sauce, olives and cocoa. (Microbes and human life, 2013) Single cell proteins are known as dried cells of microbes which are used in protein supplement shacks. They are also called “novel food” and “minifood”. The production of this requires micro-organisms which then serve as the protein source and then the substrate which is biomass which they grow on them. There are a number of both these sources that we are able to use for the production of single cell protein (SCP). The micro-organisms used belong to the following groups of Algae, Fungi and bacteria. (Slide Share, 2012)