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1.
V. M. Serebrennikov L. N. Kotova A. V. Glazunov 《Applied Biochemistry and Microbiology》2014,50(7):689-700
We investigated the effects of the oxygen supply rate on the activity of pyruvate metabolic pathways and their end products, the lactatedehydrogenase (LDH), pyruvateformiatelyase (PFL), pyruvatedehydrogenase (PDH) and acetolactatesynthase (ALS) pathways, in the Lactococcus lactis ssp. lactis bv. diacetylactis strain B2103/74. We found that this culture, apart from inactivated α-acetyldecarboxylase, also possesses a unique natural capacity to overexpress α-acetolactate (AL) up to 25–28 mM. Our search for similar properties among the diacetilicus bv. strains showed that this ability is quite rare. We identified a single additional strain, 7590 from the National Russian Collection of Industrial Microorganisms (NRCIM-7590), which displayed a similar capacity. However, unlike B2103/74, NRCIM-7590 has an active α-acetolactate decarboxylase and therefore can only produce acetoin. AL overexpression took place under conditions of intense aeration (K L a ≥ 90–120 h?1), and the composition of the medium played a decisive role in AL productivity. We found that AL overproduction is determined by a diversion of a portion of pyruvate flow from the LDH to the PDH and ALS pathways. We further found that all additional pyruvate, supplied from LDH, is utilized exclusively by the ALS pathway because of the restricted capacity of the PDH pathway. This shift in pyruvate metabolism in the B2103/74 strain, from LDH to PDH and ALS pathways, is associated with the initiation of an oxidation reaction that reduces oxygen to H2O and sequesters NADH from the LDH pathway in the process. A specific manifestation of this reaction in B2103/74 and NRCIM-7590 cultures, which results in a profound shift of the pyruvate metabolism towards the production of α-acetolactate, is due to the function of a potent oxidative system that shifts 75–80% of NADH flow from LDH to the oxidative pathway, resulting in the regeneration of NAD+. The nature of this oxidative system is not known. Based on our studies, we propose that the structure of the newly discovered oxidative system is similar to a simple transmembrane electron transport chain. 相似文献
2.
Summary Spontaneous oxidative decarboxylation of -acetolactic acid (ALA) to diacetyl has been assessed under anaerobiosis as a function of pH in water, and in fresh and filtered Lactococcus lactis spp. lactis bv. diacetylactis SD 933 fermented culture media. Whatever the reaction medium, ALA was shown to be potentially reactive, depending on the pH of medium. Diacetyl production mechanism by this strain is discussed on the basis of these kinetic data. 相似文献
3.
Summary Cell-free extracts of Leuconostoc and Lactococcus species were tested for their -acetolactate synthase and -acetolactate decarboxylase activities. In Leuconostoc mesenteroides subsp. cremoris, Leuconostoc mesenteroides subsp. mesenteroides and Leuconostoc lactis, the Km of -acetolactate synthase for pyruvate was close to 10 mM whereas it was 30 mM in Lactococcus lactis subsp. lactis biovar. diacetylactis. The Km of -acetolactate decarboxylase for -acetolactic acid was very low (0.3 mM) in Leuconostoc species in comparison to Lactococcus lactis subsp. lactis biovar. diacetylactis (60 mM). In the latter bacterium, -acetolactate decarboxylase showed a sigmoidal dependance upon -acetolactic acid and was activated by the three branchedchain amino acids: leucine, isoleucine and valine. 相似文献
4.
5.
K. J. Leenhouts A. Bolhuis A. Ledeboer G. Venema J. Kok 《Applied microbiology and biotechnology》1995,44(1-2):75-80
A plant -galactosidase gene was inserted in the expression vector pGKV259. The resulting plasmid pGAL2 consisted of the replication functions of the broad-host-range lactococcal plasmid pWV01, the lactococcal promoter P59, and the DNA sequences encoding the -amylase signal sequence from Bacillus amyloliquefaciens and the mature part of the -galactosidase from Cyamopsis tetragonoloba (guar). Lactococcus cells of strain MG1363 harbouring this vector produced the plant -galactosidase and secreted the enzyme efficiently as judged by Western blotting and activity assays. Expression levels of up to 4.3 mg extracellular -galactosidase g (dry weight) of biomass–1 were achieved in standard laboratory batch cultures. The -galactosidase produced by Lactococcus was active on the chromogenic substrate 5-bromo-4-chloro-3-indolyl -d-galactopyranoside, the trisaccharide raffinose and on the galactomannan substrate, guar gum. 相似文献
6.
Transamination is the first step in the conversion of amino acids into aroma compounds by lactic acid bacteria (LAB) used in food fermentations. The process is limited by the availability of α-ketoglutarate, which is the best α-keto donor for transaminases in LAB. Here, uptake of α-ketoglutarate by the citrate transporter CitP is reported. Cells of Lactococcus lactis IL1403 expressing CitP showed significant levels of transamination activity in the presence of α-ketoglutarate and one of the amino acids Ile, Leu, Val, Phe, or Met, while the same cells lacking CitP showed transamination activity only after permeabilization of the cell membrane. Moreover, the transamination activity of the cells followed the levels of CitP in a controlled expression system. The involvement of CitP in the uptake of the α-keto donor was further demonstrated by the increased consumption rate in the presence of l-lactate, which drives CitP in the fast exchange mode of transport. Transamination is the only active pathway for the conversion of α-ketoglutarate in IL1403; a stoichiometric conversion to glutamate and the corresponding α-keto acid from the amino acids was observed. The transamination activity by both the cells and the cytoplasmic fraction showed a remarkably flat pH profile over the range from pH 5 to pH 8, especially with the branched-chain amino acids. Further metabolism of the produced α-keto acids into α-hydroxy acids and other flavor compounds required the coupling of transamination to glycolysis. The results suggest a much broader role of the citrate transporter CitP in LAB than citrate uptake in the citrate fermentation pathway alone. 相似文献
7.
D. V. Gokhale U. S. Puntambekar A. K. Vyas S. G. Patil Dileep N. Deobagkar 《Biotechnology letters》1984,6(11):719-722
Summary An Aspergillus sp. was isolated which secreted high levels of -glucosidase in growth medium. The maximum activity(10 IU/ml of -glucosidase and 22.6 IU/ml of cellobiase) was obtained in cellulose medium supplemented with wheat bran. The pH and temperature optima for this enzyme were 4.5 and 65°C respectively.NCL Communication No. 3616 相似文献
8.
Background
Oligosaccharides containing a terminal Gal-α1,3-Gal moiety are collectively known as α-Gal epitopes. α-Gal epitopes are integral components of several medical treatments under development, including flu and HIV vaccines as well as cancer treatments. The difficulty associated with synthesizing the α-Gal epitope hinders the development and application of these treatments due to the limited availability and high cost of the α-Gal epitope. This work illustrates the development of a whole-cell biocatalyst for synthesizing the α-Gal epitope, Gal-α1,3-Lac. 相似文献9.
Dorau Robin Chen Lin Liu Jianming Jensen Peter Ruhdal Solem Christian 《Microbial cell factories》2019,18(1):1-16
With a variety of physiological and pharmacological functions, menaquinone is an essential prenylated product that can be endogenously converted from phylloquinone (VK1) or menadione (VK3) via the expression of Homo sapiens UBIAD1 (HsUBIAD1). The methylotrophic yeast, Pichia pastoris, is an attractive expression system that has been successfully applied to the efficient expression of heterologous proteins. However, the menaquinone biosynthetic pathway has not been discovered in P. pastoris. Firstly, we constructed a novel synthetic pathway in P. pastoris for the production of menaquinone-4 (MK-4) via heterologous expression of HsUBIAD1. Then, the glyceraldehyde-3-phosphate dehydrogenase constitutive promoter (PGAP) appeared to be mostsuitable for the expression of HsUBIAD1 for various reasons. By optimizing the expression conditions of HsUBIAD1, its yield increased by 4.37 times after incubation at pH 7.0 and 24 °C for 36 h, when compared with that under the initial conditions. We found HsUBIAD1 expressed in recombinant GGU-23 has the ability to catalyze the biosynthesis of MK-4 when using VK1 and VK3 as the isopentenyl acceptor. In addition, we constructed a ribosomal DNA (rDNA)-mediated multi-copy expression vector for the fusion expression of SaGGPPS and PpIDI, and the recombinant GGU-GrIG afforded higher MK-4 production, so that it was selected as the high-yield strain. Finally, the yield of MK-4 was maximized at 0.24 mg/g DCW by improving the GGPP supply when VK3 was the isopentenyl acceptor. In this study, we constructed a novel synthetic pathway in P. pastoris for the biosynthesis of the high value-added prenylated product MK-4 through heterologous expression of HsUBIAD1 and strengthened accumulation of GGPP. This approach could be further developed and accomplished for the biosynthesis of other prenylated products, which has great significance for theoretical research and industrial application. 相似文献
10.
The culture parameters required for optimum production of extracellular α-amylase by the thermophilicBacillus coagulans are described. The optimum pH, temperature and incubation period for amylase production were 7, 50°C and 48 h, respectively.
Age of inoculum (48 h) and its level, (2%) were critical for maximum amylase yield. The enzyme secretion was high in rice
starch and beef extract as compared to other carbon and nitrogen sources tested. The addition of mustard oil cake (1%) and
agitation at 1.7 Hz resulted in an enhancement of α-amylase secretion. 相似文献
11.
H. E. M. Mc Mahon C. T. Kelly W. M. Fogarty 《Applied microbiology and biotechnology》1997,48(4):504-509
The α-amylase of Streptomyces sp. IMD 2679 was subject to catabolite repression. Four different growth rates were achieved when the organism was grown
at 40 °C and 55 °C in the presence and absence of cobalt, with an inverse relationship between α-amylase production and growth
rate. Highest α-amylase yields (520 units/ml) were obtained at the lowest growth rate (0.062 h−1), at 40 °C in the absence of cobalt, while at the highest growth rate (0.35 h−1), at 55 °C in the presence of cobalt, α-amylase production was decreased to 150 units/ml. As growth rate increased, the rate
of specific utilisation of the carbon source maltose also increased, from 46 to 123 μg maltose (mg biomass)−1 h−1. The pattern and levels of α-glucosidase (the enzyme degrading maltose) detected intracellularly in each case, indicate that
growth rate effectively controls the rate of feeding of glucose to the cell, and thus catabolite repression.
Received: 17 February 1997 / Received revision: 29 April 1997 / Accepted: 11 May 1997 相似文献
12.
Summary Two extracellular -glucosidases (EC 3.2.1.20, -D-glucoside glucohydrolase) of the alkalophilic bacterium,Bacillus sp. NCIB 11203, were separated, purified and partially characterised. Resolution of the system into two separate enzymes was achieved by fractionation with (NH4)2SO4 and chromatography on DEAE-Biogel A. The first of these activities, an -glucosidase, hydrolysed p-nitrophenyl--D-glucopyranoside preferentially and had minor activity on isomaltose and isomaltotriose. The second enzyme was a maltase and displayed highest activity on maltose and maltotriose and some activity on p-nitrophenyl--D-glucopyranoside. 相似文献
13.
Decarboxylation rates for a series of C-3 to C-6 α-keto acids were determined in the presence of resting cells and cell-free extracts of Streptococcus lactis var. maltigenes. The C-5 and C-6 acids branched at the penultimate carbon atom were converted most rapidly to the respective aldehydes in the manner described for α-carboxylases. Pyruvate and α-ketobutyrate did not behave as α-carboxylase substrates, in that O2 was absorbed when they were reacted with resting cells. The same effect with pyruvate was noted in a nonmalty S. lactis, accounting for CO2 produced by some “homofermentative” streptococci. Mixed substrate reactions indicated that the same enzyme was responsible for decarboxylation of α-ketoisocaproate and α-ketoisovalerate, but it appeared unlikely that this enzyme was responsible for the decarboxylation of pyruvate. Ultrasonic disruption of cells of the malty culture resulted in an extract inactive for decarboxylation of pyruvate in the absence of ferricyanide. Dialyzed cell-free extracts were inactive against all keto acids and could not be reactivated. 相似文献
14.
Marcel J. Teunissen Daniëlle H. T. P. Lahaye Jos H. J. Huis in 't Veld Godfried D. Vogels 《Archives of microbiology》1992,158(4):276-281
An extracellular -glucosidase (EC 3.2.2.21) from the anaerobic fungus Piromyces sp. strain E2 was purified. The enzyme is a monomer with a molecular mass of 45 kDa and a pI of 4.15. The enzyme readily hydrolyzes p-nitrophenyl--d-glycoside, p-nitrophenyl--d-fucoside, cellobiose, cellotriose, cellotetraose and cellopentaose but is not active towards Avicel, carboxymethylcellulose, xylan, p-nitrophenyl--d-galactoside and p-nitrophenyl--d-xyloside. To cleave p-nitrophenyl--d-glucoside the maximum activity is reached at pH 6.0 and 55°C, and the enzyme is stable up to 72 h at 40°C. Activity is inhibited by d-glucurono--lactone, cellobiose, sodium dodecyl sulfate, Hg2+ and Cu2+ cations. With p-nitrophenyl--d-glycoside, p-nitrophenyl--d-fucoside, and. cellobiose as enzyme substrates, the K
m and V
max balues are 1.5 mM and 25.5 IU·mg-1, 1.1. mM and 133 IU·mg-1, and 0.05 mM and 55.6 IU·mg-1, respectively. 相似文献
15.
《Enzyme and microbial technology》1987,9(1):53-56
Bacillus subtillis ATCC 21770 was entrapped in a carrageenan gel, especially formulated for immobilization. Bacterial growth and α-amylase (1,4-α-d-glucan glucanohydrolase EC 3.2.1.1) production were tested. The bead suspensions were submitted to two aeration modes, one consisting of bubbling air into a round flask, the other involving sparging of air into an airlift fermenter. The latter system, which produces microbubbles, gave 40–70% increase in enzyme production over the former and a doubling of bacterial density within the beads was measured. The use of CaCl2instead of KCl as polymerization agent led to a better yield of α-amylase. 相似文献
16.
Avutu S. Reddy Michael L. Nuccio Lisa M. Gross Terry L. Thomas 《Plant molecular biology》1993,22(2):293-300
The enzyme 6-desaturase is responsible for the conversion of linoleic acid (18:2) to -linolenic acid (18:3). A cyanobacterial gene encoding 6-desaturase was cloned by expression of a Synechocystis genomic cosmid library in Anabaena, a cyanobacterium lacking 6-desaturase. Expression of the Synechocystis 6-desaturase gene in Anabaena resulted in the accumulation of -linolenic acid (GLA) and octadecatetraenoic acid (18:4). The predicted 359 amino acid sequence of the Synechocystis 6-desaturase shares limited, but significant, sequence similarity with two other reported desaturases. Analysis of three overlapping cosmids revealed a 12-desaturase gene linked to the 6-desaturase gene. Expression of Synechocystis 6-and 12-desaturase in Synechococcus, a cyanobacterium deficient in both desaturases, resulted in the production of linoleic acid and -linolenic acid. 相似文献
17.
A gene involved in the production of medium-chain α-olefins was identified in the cyanobacterium Synechococcus sp. strain PCC 7002. The gene encodes a large multidomain protein with homology to type I polyketide synthases, suggesting a route for hydrocarbon biosynthesis from fatty acids via an elongation decarboxylation mechanism. 相似文献
18.
An α-glucosidase (HaG) with the following unique properties was isolated from Halomonas sp. strain H11: (i) high transglucosylation activity, (ii) activation by monovalent cations, and (iii) very narrow substrate specificity. The molecular mass of the purified HaG was estimated to be 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HaG showed high hydrolytic activities toward maltose, sucrose, and p-nitrophenyl α-D-glucoside (pNPG) but to almost no other disaccharides or malto-oligosaccharides higher than trisaccharides. HaG showed optimum activity to maltose at 30°C and pH 6.5. Monovalent cations such as K(+), Rb(+), Cs(+), and NH(4)(+) increased the enzymatic activity to 2- to 9-fold of the original activity. These ions shifted the activity-pH profile to the alkaline side. The optimum temperature rose to 40°C in the presence of 10 mM NH(4)(+), although temperature stability was not affected. The apparent K(m) and k(cat) values for maltose and pNPG were significantly improved by monovalent cations. Surprisingly, k(cat)/K(m) for pNPG increased 372- to 969-fold in their presence. HaG used some alcohols as acceptor substrates in transglucosylation and was useful for efficient synthesis of α-d-glucosylglycerol. The efficiency of the production level was superior to that of the previously reported enzyme Aspergillus niger α-glucosidase in terms of small amounts of by-products. Sequence analysis of HaG revealed that it was classified in glycoside hydrolase family 13. Its amino acid sequence showed high identities, 60%, 58%, 57%, and 56%, to Xanthomonas campestris WU-9701 α-glucosidase, Xanthomonas campestris pv. raphani 756C oligo-1,6-glucosidase, Pseudomonas stutzeri DSM 4166 oligo-1,6-glucosidase, and Agrobacterium tumefaciens F2 α-glucosidase, respectively. 相似文献
19.
Brewer's spent grain (BSG) was used as a solid substrate for the production of α-amylase by Bacillus sp. KR-8104 in a submerged fermentation system. The production of α-amylase was maximized through statistical optimization of the BSG concentration and incubation time using the Doehlert experimental design. The highest tested amount of BSG (5%, w/v) in the optimization process resulted in a 5.1-fold enhancement of the response. Subsequently, we studied the role of the water-soluble and -insoluble fractions of BSG in the production of α-amylase. The results revealed that whole BSG had a greater effect on the production of α-amylase than each fraction had separately. Finally, when we examined the potential of BSG to replace the constituents of a medium formula, we observed that simultaneously adding BSG, omitting dextrin, and reducing the other ingredients concentration in the culture medium improved the production of α-amylase and made the production process more economical. 相似文献
20.
Tao Jiang Mengmeng Huang Hao He Jian Lu Xiangshan Zhou 《Preparative biochemistry & biotechnology》2016,46(6):620-627
Geobacillus sp. 4j, a deep-sea high-salt thermophile, was found to produce thermostable α-amylase. In this work, culture medium and conditions were first optimized to enhance the production of thermostable α-amylase by statistical methodologies. The resulting extracellular production was increased by five times and reached 6.40?U/ml. Then, a high-temperature batch culture of the thermophile in a 15?l in-house-designed bioreactor was studied. The results showed that a relatively high dissolved oxygen (600?rpm and 15?l/min) and culture temperature of 60°C facilitated both cell growth and α-amylase production. Thus, an efficient fermentation process was established with initial medium of pH 6.0, culture temperature of 60°C, and dissolved oxygen above 20%. It gave an α-amylase production of 79?U/ml and productivity of 19804?U/l·hr, which were 10.8 and 208 times higher than those in shake flask, respectively. This work is useful for deep-sea high-salt thermophile culture, where efforts are lacking presently. 相似文献