Browsing by Subject "protease"

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  • Bäcklund, Kirsi (Helsingfors universitet, 2013)
    Boreal forest soil contains large nitrogen resources. Most of the nitrogen is bound to humified material and proteins. However, plants can directly utilize only simple nitrogen compounds as ammonium, nitrate and simple amino acids. The amount of these simple nitrogen compounds is very small in forest soil. Trees form ectomycorrhizal symbiosis with a large number of fungi. Ectomycorrhizal fungi receive carbon compounds from host trees and trees receive nutrients from fungi. Some ectomycorrhizal fungi produce proteases which are enzymes that hydrolyze proteins. This symbiosis might be important in utilizing the large nitrogen resources in forest soils and supplying nitrogen to host plants. The main aim of this study was to measure if ectomycorrhizas produce proteases in the field and to find out if the mycorrhizal protease activities change during the year. The aim was also to find out if proteolytic activity is found in the soil fluid and to see if the activities in soil fluid are related to mycorrhizal activities. Mycorrhizal fungi were isolated to form pure cultures and their protease activities were measured and species were identified. As background information mycorrhizal, nonmycorrhizal and dead root tips were calculated to see how their amounts change during the year. Also different kinds of protease producing mycorrhizal morphotypes were calculated. Samples were taken in Hyytiälä Forestry Field Station in Central Finland monthly from March to October. Fifteen soil cores were collected each month. If possible, eight ectomycorrhizas were randomly picked from the pine roots in the humus layer of each soil core. Protease activities were measured from the ectomycorrhizas and from the soil solution obtained from the homogenized humus layer using Protease Fluorescent Detection Kit (PF0100-1KT, SIGMA). Detection limit of the kit was enzyme activity that is equivalent to 5 ng of trypsin control activity. Results were read from the trypsin standard curve so the protease activities of the samples are equivalent to fluorescence of certain amount of trypsin control. Pure cultures were isolated to Hagem’s agar plates and species were identified by doing PCR from the ITS gene region and sequencing. Root tip and morphotype calculations were done by using a microscope. 12 % of all ectomycorrhizal root tips produced proteases. The smallest activities couldn’t be detected because of the detection limit of the kit. Ectomycorrhizal protease production varied between 0–12 ?g/m2 of soil. Statistically non-significant protease production peaks were observed in spring and autumn. The protease activity of the soil fluid varied mainly between 0–200 ?g/m2 of soil. The highest activity was detected in the frozen soil in March, when the variation was large and an average of 800 ?g of protease was measured per m2 of soil. The protease activities in the soil fluids were not related to the protease activities of the ectomycorrhizas. 17 pure cultures were achieved. Some of them couldn’t be identified to species level. Part of the pure cultures produced proteases. Number of mycorrhizal root tips was large in spring, decreasing in early summer and increasing again towards autumn. About half of the mycorrhizal morphotypes produced proteases. The most important conclusions are that ectomycorrhizal fungi produce proteases in the field and a lot of protease activity is also found in the soil fluid. The used method is sensitive and suitable for measuring protease activities directly from mycorrhizal root tips and soil fluid. Seasonal variation in the protease activities may occur both in mycorrhizas and in soil fluid.
  • Hussain, Nazar; Ran, Li; Takala, Timo; Tariq, Muhammad; Zaidi, Arsalan; Saris, Per (2021)
    Lacticaseibacillus rhamnosus GG (LGG) is the most studied probiotic bacterium in the world. It is used as a probiotic supplement in many foods, including various dairy products. However, LGG grows poorly in milk, as it neither metabolizes the main milk carbohydrate lactose nor degrades the major milk protein casein effectively. In this study, we made L rhamnosus GG lactose and protease positive by conjugation with the dairy Lactococcus lactis strain NCDO 712 carrying the lactose-protease plasmid pLP712. A lactose-hydrolyzing transconjugant colony was obtained on agar containing lactose as the sole source of carbohydrates. By microscopic analysis and PCR with LGG- and pLP712-specific primers, the transconjugant was confirmed to have originated from LGG and to carry the plasmid pLP712. The transconjugant was named L. rhamnosus LAB49. The isolation of plasmids revealed that not only pLP712 but also other plasmids had been transferred from L lactis into LGG during conjugation. With plasmid-specific PCR primers, four additional lactococcal plasmids were detected in LAB49. Proteolytic activity assay and SDS-PAGE analysis verified that L rhamnosus LAB49 effectively degraded beta-casein. In contrast to its parental strain, LGG, the ability of LAB49 to metabolize lactose and degrade casein enabled strong and fast growth in milk. As strains with new properties made by conjugation are not regarded as genetically modified organisms (GM05), L. rhamnosus LAB49 could be beneficial in dairy fermentations as a probiotic starter culture. IMPORTANCE Probiotic strain Lacticaseibacillus rhamnosus GG (LGG) is widely sold on the market as a probiotic or added as a supplement in dairy foods because of its benefits in human health. However, due to the deficiency of lactose and casein utilization, LGG does not grow well in milk. On the other hand, lactose intolerance and cow's milk protein allergy are the two major problems related to milk consumption. One option to help with these two conditions is the use of probiotic or lactose- and casein-hydrolyzing bacteria in dairy products. The purpose of this study was to equip LGG with lactose/casein-hydrolyzing ability by bacterial conjugation. As a result, we generated a non-GMO LGG derivative with improved properties and better growth in milk.
  • Pöntinen, Anna; Aalto-Araneda, Mariella; Lindström, Miia; Korkeala, Hannu (2017)
    Listeria monocytogenes is one of the most heat-resistant non-sporeforming food-borne pathogens and poses a notable risk to food safety, particularly when mild heat treatments are used in food processing and preparation. While general heat stress properties and response mechanisms of L. monocytogenes have been described, accessory mechanisms providing particular L. monocytogenes strains with the advantage of enhanced heat resistance are unknown. Here, we report plasmidmediated heat resistance of L. monocytogenes for the first time. This resistance is mediated by the ATP-dependent protease ClpL. We tested the survival of two wildtype L. monocytogenes strains-both of serotype 1/2c, sequence type ST9, and high sequence identity-at high temperatures and compared their genome composition in order to identify genetic mechanisms involved in their heat survival phenotype. L. monocytogenes AT3E was more heat resistant (0.0 CFU/ml log(10) reduction) than strain AL4E (1.4 CFU/ml log(10) reduction) after heating at 55 degrees C for 40 min. A prominent difference in the genome compositions of the two strains was a 58-kb plasmid (pLM58) harbored by the heat-resistant AT3E strain, suggesting plasmid-mediated heat resistance. Indeed, plasmid curing resulted in significantly decreased heat resistance (1.1 CFU/ml log(10) reduction) at 55 degrees C. pLM58 harbored a 2,115-bp open reading frame annotated as an ATP-dependent protease (ClpL)-encoding clpL gene. Introducing the clpL gene into a natively heat-sensitive L. monocytogenes strain (1.2 CFU/ml log(10) reduction) significantly increased the heat resistance of the recipient strain (0.4 CFU/ml log(10) reduction) at 55 degrees C. Plasmid-borne ClpL is thus a potential predictor of elevated heat resistance in L. monocytogenes. IMPORTANCE Listeria monocytogenes is a dangerous food pathogen causing the severe illness listeriosis that has a high mortality rate in immunocompromised individuals. Although destroyed by pasteurization, L. monocytogenes is among the most heat-resistant non-spore-forming bacteria. This poses a risk to food safety, as listeriosis is commonly associated with ready-to-eat foods that are consumed without thorough heating. However, L. monocytogenes strains differ in their ability to survive high temperatures, and comprehensive understanding of the genetic mechanisms underlying these differences is still limited. Whole-genome-sequence analysis and phenotypic characterization allowed us to identify a novel plasmid, designated pLM58, and a plasmid-borne ATP-dependent protease (ClpL), which mediated heat resistance in L. monocytogenes. As the first report on plasmid-mediated heat resistance in L. monocytogenes, our study sheds light on the accessory genetic mechanisms rendering certain L. monocytogenes strains particularly capable of surviving high temperatures-with plasmid-borne ClpL being a potential predictor of elevated heat resistance.
  • Beyer, Hannes M.; Virtanen, Salla; Aranko, A. Sesilja; Mikula, Kornelia M.; Lountos, George T.; Wlodawer, Alexander; Ollila, O.H. Samuli; Iwai, Hideo (2020)
    Protein splicing catalyzed by inteins utilizes many different combinations of amino-acid types at active sites. Inteins have been classified into three classes based on their characteristic sequences. We investigated the structural basis of the protein splicing mechanism of class 3 inteins by determining crystal structures of variants of a class 3 intein from Mycobacterium chimaera and molecular dynamics simulations, which suggested that the class 3 intein utilizes a different splicing mechanism from that of class 1 and 2 inteins. The class 3 intein uses a bond cleavage strategy reminiscent of proteases but share the same Hedgehog/INTein (HINT) fold of other intein classes. Engineering of class 3 inteins from a class 1 intein indicated that a class 3 intein would unlikely evolve directly from a class 1 or 2 intein. The HINT fold appears as structural and functional solution for trans-peptidyl and trans-esterification reactions commonly exploited by diverse mechanisms using different combinations of amino-acid types for the active-site residues.
  • Jayachandra, Yaradoddi A.; Sulochana, M. B.; Merja, Kontro H.; Parameshwar, A. B.; Dayanand, Agsar (2020)
    Many halophiles were considered to be extremophiles due to their inborn industrial potentials and tolerance to hostile environmental conditions. The isolated halophilic bacteria described in the present study are not only grown at environmentally adverse conditions, also they can be able to produce bioactive molecules. Among the isolated strains, Oceanobacillus iheyensis strain JAS12 and Salinicoccus roseus strain JS20 are known for the unique biotechnological applications. The isolate Oceanobacillus sp. grows well at 35-55 degrees C (optimum 45 degrees C) and pH 6 to 12 (maximum growth at pH 8), interestingly the strain could hydrolyze casein, starch and gelatin. The G+C content was 40.2 mol % and the major fatty acids are iso-15:0: 30.52%, primary-C15: 0 (29.29 %), iso-14:0 (16.15%) anteiso-C17: 0 (4.03%). Another isolate was Salinicoccus sp. JS20 The DNA G+C content was 50.4 mol % and the major fatty acids are anteiso-C15: 0 (26.23%), iso15:0, (17.62%)Y, 16:0 (11.5%), anteiso-C17: 0 (7.7 %), iso- C16: 0 (10.20 %), iso-17:0: (5.43%) and iso-C14: 0 (3.97 %). These isolates are also producers of many extracellular enzymes such as protease, amylase, inulinases, gelatinase and beta-fructofurinosidase above the optimal conditions. Oceanobacillus sp. JAS12 16S rRNA gene sequence similarity is 99% similar to the reported genera. Salinicoccus sp. JS20 indicated 96% 16S rRNA sequence similarity with near species Salinicoccus genus, thus, they were found to be novel concerning to their genetic makeup and biochemical features.
  • Koivunen, Erja; Talvio, Eija; Valkonen, Eija; Tupasela, Tuomo; Tuunainen, Petra; Valaja, Jarmo (2016)
    The aim was to study the effects of dietary pea inclusion and the addition of Avizyme 1200 -enzyme cocktail on broiler performance, intestinal viscosity and organoleptic quality of meat. The experimental design was a 4 x 2 factorial, the factors being dietary pea inclusion (0, 150, 300 and 450 g kg(-1)) in the diets fed from day 9 to day 38 and the addition of Avizyme 1200 enzyme cocktail including amylase, protease and xylanase during the entire experiment. The growth of birds improved (p 0.05). In conclusion, 450 g kg(-1) peas can be used in the broiler grower diets without negative effects on the bird performance. The use of enzyme cocktail improves bird performance.