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Julius Semyonov
Julius Semyonov

A Acid Factory Download ((FULL))



Acid Factory is one of the best new collection games and platform games our team is very happy that we have managed to add into the Miniclip Games category from our website, which we know is one of the best new categories that we have created for our website recently, so we have no doubt at all that you are going to be very happy with all of the games that you will manage to find here. We will now proceed to explain what you have to do in this one, so make sure you carefully read the next lines of the description! Collect the batteries to power up the portal, to get through it to the next level. Do not touch the acid, since it kills you, and also try your best to avoid the monsters as well. Pick up a gun and kill monster to score more points, with the more dangerous the monster is, the more points you are rewarded with. Try to get out of each level as quickly as possible, because the more air you save, the more points you get. Obviously, you are going to use the arrow keys to move around, and the space bar to use the gun if necessary. That's about it, so we want to wish you all good luck, in hopes you have a great time with this game!




A Acid Factory Download


Download File: https://www.google.com/url?q=https%3A%2F%2Fjinyurl.com%2F2u31zB&sa=D&sntz=1&usg=AOvVaw146ZUpuWDAwCEQrmmn9iRK



Envirofacts Provides all publicly available data collected by the GHGRP in a searchable, downloadable format for facilities. This includes GHG data and much of the underlying data facilities use to determine GHG values and other reported data elements in 32 industry types.


Facility Level Information on GreenHouse Gases Tool (FLIGHT) An interactive website with mapping features to identify GHGRP facilities by location, name, industry type, and other criteria. FLIGHT can also generate and download customized graphics (pie charts, trend lines, etc.) and facility lists.


The Adipic acid tool allows the N2O emissions from adipic acid production to be estimated. It requires data on the amount of adipic acid produced. The tool also calculates any emissions reductions associated with the use of emissions control technologies.


The Nitric acid tool allows the N2O emissions from nitric acid production to be estimated. It requires data on the amount of nitric acid produced. The tool also calculates any emissions reductions associated with the use of emissions control technologies.


From fizzy tablets with cleaning power and gently rejuvenating face washes to fruity cough drops and palatable cough syrups, malic acid is used in many medical, personal care, and cosmetic applications as a functionally versatile and flavor-enhancing ingredient.


Kojic acid (5-Hydroxy-2-(hydroxymethyl)-4-pyrone) is one of the major secondary metabolites in Aspergillus oryzae. It is widely used in food, pharmaceuticals, and cosmetics. The production cost, however, is too high for its use in many applications. Thus, an efficient and cost-effective kojic acid production process would be valuable. However, little is known about the complete set of genes for kojic acid production. Currently, kojic acid is produced from glucose. The efficient production of kojic acid using cellulose as an inexpensive substrate would help establish cost-effective kojic acid production.


A kojic acid transcription factor gene over-expressing the A. oryzae strain was constructed. Three genes related to kojic acid production in this strain were transcribed in higher amounts than those found in the wild-type strain. This strain produced 26.4 g/L kojic acid from 80 g/L glucose. Furthermore, this strain was transformed with plasmid harboring 3 cellulase genes. The resultant A. oryzae strain successfully produced 0.18 g/L of kojic acid in 6 days of fermentation from the phosphoric acid swollen cellulose.


Kojic acid was produced directly from cellulose material using genetically engineered A. oryzae. Because A. oryzae has efficient protein secretion ability and secondary metabolite productivity, an A. oryzae-based cell factory could be a platform for the production of various kinds of bio-based chemicals.


Kojic acid (5-Hydroxy-2-(hydroxymethyl)-4-pyrone) is widely used in the food industry, pharmaceutical industry, and in cosmetics[1, 2]. Furthermore, kojic acid is also used as a building block for biodegradable plastics[3, 4]. Plastics have phenolic hydroxyl groups that are derived from kojic acid. Thus, it has some unique properties. Futamura et al.[5] reported that the production cost of kojic acid is about $10/kg. It must be decreased to less than $2/kg before it could be used in many applications. Thus, an efficient and cost-effective kojic acid production process would be desirable.


Kojic acid is one of the major secondary metabolites in A. oryzae. In the 100 years since it was discovered, there has been no elucidation of the entire pathway of kojic acid biosynthesis in A. oryzae. Recently, some genes regarding the production of kojic acid in A. oryzae were reported[9]. According to the report, 14 genes from AO090113000132 to AO090113000145 including a transcription factor gene (kojR; AO090113000137), an enzyme gene (kojA, AO090113000136), and a transporter gene (kojT AO090113000138) form a cluster. These genes are engaged in kojic acid production. The kojR gene encodes a fungal-specific Zn(II)2Cy6 transcription factor that is located between kojA (upstream 743 bp) and kojT (downstream 383 bp). The kojR regulates the transcription of kojA and kojT. A strain with a disrupted transcription factor gene could not produce kojic acid at all[10]. Thus, kojR seems to be a key factor in kojic acid production.


The goal of the present study was to construct an A. oryzae-based cell factory for direct kojic acid production from cellulose. First, A. oryzae over-expressing the transcription factor gene kojR was constructed, and kojic acid production from glucose and starch by the resultant strain was carried out. Then, A. oryzae co-expressing the transcription factor and 3-types of cellulase genes was constructed. Finally, direct kojic acid production from cellulose by the resultant strain was carried out.


The BGL1 gene from A. aculeatus expressing plasmid pIS1-BGL1 was constructed as follows. The DNA fragment encoding the secretion signal from A. oryzae cutinase, the 28 amino acids from the N-terminal region of Rhizopus oryzae lipase, and the A. aculeatus BGL gene were amplified by PCR using the primers sCutL-F and sCutL-R, N28-F and N28-R, and BGL-F and BGL-R, respectively, from the genome DNA of A. oryzae, pISI-GFP[14], and pδU-PGAGBGL[15], respectively. These fragments were simultaneously inserted into the AscI/NotI site of the plasmid pISI using an In-Fusion HD Cloning Kit. The resultant plasmid was named pISI-BGL.


The transcription level of Kojic-acid-producing genes (kojR, kojA, kojT) was quantified by real-time PCR. Quantitative real-time PCR was performed using an Mx3005P Real-Time QPCR System (Agilent Technologies, Santa Clara, CA, USA) with a Thunderbird SYBR qPCR Mix (Toyobo). The normalized transcription level was calculated using the standard curve method with β-tubulin as the house-keeping gene[20]. All primers used for real-time PCR analysis are summarized in Table 2.


The kojic acid concentration was determined using a colorimetric method described previously[21]. The total sugar concentration was determined by a colorimetric phenol-sulfuric acid method described previously[22]. The glucose concentration was determined using a Wako Glucose CII-Test kit (Wako Pure Chemical Industries, Osaka, Japan).


To confirm the effect of the over-expression of the transcription factor for kojic acid production, the kojic acid productivity from the transcription factor over-expressing strain NSPlD1/pIS1-kojR from glucose was evaluated. As shown in Figure 1 the wild-type strain produced 16.4 g/L of kojic acid after 14 days of fermentation. By contrast, the transcription factor over-expressing strain NSPlD1/pIS1-kojR produced 26.4 g/L of kojic acid after 14 days of fermentation and it was 1.6-fold higher than that of the wild-type strain.


Time course of kojic acid production from glucose. Triangles, glucose; squares, kojic acid; open symbols, wild-type strain; and, closed symbols, transcription factor over-expressing strain NSPlD1/pIS1-kojR. The data represent the averages from three independent experiments (error bars represent SE).


To confirm the effect of the over-expression of transcription factor at the level of the kojic-acid-producing genes, the transcription level of the transcription factor, the kojic-acid-producing enzyme, and the transporter gene were evaluated by real-time PCR. As shown in Figure 2, the transcription factor of the over-expressing strain NSPlD1/pIS1-kojR showed a 1.8, 1.8, and 5.4-fold increases in the transcription level of transcription factor, enzyme, and transporter gene, respectively, compared with the wild-type strain.


Transcription levels of kojic acid producing genes. (A) Transcription factor gene, (B) enzyme gene, (C) transporter gene. The data represent the averages from three independent experiments (error bars represent SE).


Since A. oryzae can assimilate starch as a sole carbon source intrinsically, direct kojic acid production from starch using the transcription factor over-expressing strain NSPlD1/pIS1-kojR was carried out. As shown in Figure 3, the wild-type strain produced 6.2 g/L of kojic acid after 18 days of fermentation. In contrast, the transcription factor over-expressing strain produced 19.4 g/L of kojic acid after 18 days of fermentation, i.e. 3.1-fold gain.


Time course of kojic acid production from starch. Triangles, total sugar; squares, kojic acid; open symbols, wild-type strain; and, closed symbols, transcription factor over-expressing strain NSPlD1/pIS1-kojR. The data represent the averages from three independent experiments (error bars represent SE).


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