| Malachite green is a triphenylmethane dye which has been widely used as an antibacterial agent and insecticide in aquaculture since 1930s and also has been widely used as a colorant in industry, such as food, health and textile. In recent years, it is found that malachite green is a potential genotoxic, carcinogenic and chromosomal mutagenic agent arising from environmental pollution and has increased great concerns about the food safety issues. Biological degradation, being inexpensive and eco-friendly, is considered as a valuable method for the removal of many toxic pollutants from environment. So it is an important research item to isolate microorganism degrading malachite green quickly, to clarify the degradation mechanisms, and to explore the potential of microbial resource. The propose of this study was to isolate the efficient malachite green-degrading bacteria, investigate the growth and malachite green degrading characteristics of the strain in different environments, and provide the theoretical bases for the bioremediation of environment polluted with malachite green. At the same time, cloning and expression of the malachite green degrading gene from the strain would lay the foundation for researching the gene function and improving degrading malachite green capacity by construction of the gene engineering strain.From the samples taken from malachite green-contaminated aquatic breeding pool, twenty six malachite green-grading bacterial strains were also isolated using selective culture medium comprised of malachite green as sole carbon source. The highest effective strain of them, named MDB-1, was identified as Pseudomonas stutzeri species based on morphological, physiological, and biochemical tests combined with the comparative analysis of 16S rRNA gene sequence. Comparative studies were performed on the phylogenetic relationship of the above 26 strains based on their 16S rRNA gene sequence.The results revealed that these twenty-six malachite green-degrading strains mainly come from 6 genuses:Pseudomonas, Staphylococcus, Enterobater, Alcaligenes, Microococcus and Psychrobacter.The optimal growth conditions of strain MDB-1 were investigated. The results showed the optimal growth temperature and pH of strain MDB-1 were 30℃ and 7.0, respectively. The oxygen availability significantly affects the growth of strain MDB-1. The strain grew better when the liquid volume in 250 ml flasks was less than 50 ml. MDB-1 was also able to grow in NaCl with the concentration from 0 to 3%. However, the optimal NaCl concentration is 1% for its growth. For strain MDB-1, starch and dextrin were the best and the better carbon source. Yeast extract was best nitrogen source for strain MDB-1 growth when glucose was used as carbon source. Generally, inorganic nitrogen was not an optimal nitrogen source for strain MDB-1. When strain MDB-1 grew in the fermentation medium, the lag phase last from 0-6 h, the exponential phase from 6-12 h, the stationary phase from 12-20 h and the decline phase was observed after 20 h.The malachite green degrading properties of strain MDB-1 were also demonstrated. Strain MDB-1 could grow utilizing malachite green as sole carbon source. It could degrade 10 mg·L-1 malachite green in 48 h. It was also able to degrade other triphenylmethane dyes like Crystal Violet and Basic Fuchsin. The appropriate malachite green concentration for MDB-1 degrading was below 50 mg·L-1 and the malachite green in the concentration rang from 1 to 5 mg·L-1 can be near completely degraded by MDB-1. For MDB-1 degrading malachite green, the optimum temperature and pH range was 25℃～30℃ and 8.0～9.0 respectively and the optimum medium volume in the 250 mL flask is 25 mL, while the appropriate inoculum size is 4.5×10 cell (3% v/v). Adding carbon source such as lactose, dextrin, starch, maltose, sucrose, fructose as well as nitrogen source such as yeast extract, peptone, beef extract and (NH4)SO4, considerable enhanced the malachite green degradation ability of strain MDB-1. The strain MDB-1 can also degrade the malachite green added repeatedly (more than 5 repeats were trailed).The primers were constructed based on the published sequences of the triphenylmethane reductase gene. The triphenylmethane reductase gene tmr2, comprised of 864 bp encoding 288 amino acids, was cloned by PCR from strain MDB-1. At the level of gene, the comparison of tmr2 gene sequence from MDB-1 with the relative sequences in GeneBank revealed that nine base points of triphenylmethane reductase gene are different among five degrading strains while the similarities between them were from 99% to 100%.At the level of amino acid, five points of triphenylmethane reductase are different in five degrading strains, while the similarities were also from 99% to 100%. The five degrading strains can be divided into two subtypes i.e. A and B, according to the differences in genes and amino acids. The 1600bp upstream segment and 3000bp downstream segment of tmr2 gene was cloned by SEFA-PCR from the MDB-1 and sequenced. According to the analysis that there was a transposase in the downstream of tmr2 gene sequence and the content of G+C% have significant differences between the tmr2 gene and the 16S rRNA sequences in three malachite green-grading bacteria. We deduced that tmr2 gene was likely to transfer among the malachite green-grading bacteria.The tmr2 gene encoding triphenylmethane reductase from the strain MDB-1 was ligated to pET 29a expression plasmid vector, and the expression of the gene was obtained after transformating the expression plasmid into strain E. coli BL21. SDS-PAGE analysis showed that the molecular weight of a single subunit of proteins encoded by tmr2 is 30 kd. The expressive protein was triphenylmethane reductase which can degrade malachite green and crystal violet quickly and depended on the coenzyme NADH (NADPH). The expressive protein was purified and characterized. First, the enzymatic reaction system was established:0.1 mol·L-1 PBS (pH7.5) 969 μL, 5mg·mL-1 MG 6 μL, 5mmol·L-1 NADH 20 μL, the purified enzyme 5 μL. The reaction was controlled in 1 min and terminated with 1mL CH2Cl2, then determined immediately OD622 of the malachite green in dissolved CH2Cl2. An enzyme activity unit (U) was defined as:at pH 7.5 and temperature 50℃, the amount of enzyme that catalyzed the reduction of 1μmol of malachite green per min. The research results of enzyme characterization showed that the optimum reaction temperature and pH was 50℃ and 7.5, respectively; the thermostability of the enzyme was well and the enzyme activity lost slowly below 60℃. The enzyme activity was highly maintained at pH 5.0-8.0, while 13 kinds of metal ions i.e.Ag+, Cu2+, Ca2+, Fe2+, Zn2+, inhibited the enzyme activity at an increased concentrations of ions, however Ca2+, Zn2+, Mg2+, Li+ showed a little impact on the enzyme activity.The tmr2 gene from the MDB-1 strain was ligated into the expression plasmid vector pPICZaA, and the expression of tmr2 gene was gained by transfer of the expression plasmid into Pichia pastoris GS115 strain. The protein expressed was separated easily from the broth because the major part of the expressed protein was secreted in supernatant of the broth. The optimum high-density fermentation parameters were defined by single-factor experiment:BMGY medium was used for culturing inoculum. When OD600 reached about 12, the broth was centrifuged and the precipitant was resuspended with 1/10 of the broth volume BMMY, and then induced with 2.5% methanol at pH 6.0. The enzyme activity rose to max in 72 h. In above conditions, the enzyme activity in the supernatant reached up to 134.9U which increased 3.6 times than before the optimization. |
PDF Full Text Request