Characterization Of ASPERGILLUS AW AMORI Y8 Xylanase And Its Application As A Feed Additive In Pilot Trials

Xylanases are a group of enzymes that break down xylans into xylooligosaccharides, xylose and arabinose, and thus are of high potential for use in feed, paper making and food industry. In this study, a mutant strain, Aspergillus awamori Y8, was obtained by screening out from soil isolates and subsequent breeding based on chemical and physical mutation. The mutant was featured with simultaneous production of xylanase, β-glucanase, cellulase and acidophilic protease at conspicuous levels. Of those, A. awamori Y8 xylanase, the most representative enzyme, was investigated with emphasis on optimization of fermentation conditions for high production, characterization of enzymatic features, and practical application of xylanase-based preparation in pilot trials.Screening and mutation of xylanase-producing strains. The ingredients of a medium developed for screening of xylanase-producing strain in this study included birchwood xylan 10.0 g/L, yeast extract 0.5 g/L, tryptone 0.5 g/L, and agar 15.0 g/L plus penicillin G (1610 U/mg) 0.25 g/L and streptomycin sulfate (761 U/mg) 0.25 g/L. With this method, the sizes of clear zones surrounding colonies in Petri dishes were directly proportionate to their xylanase activities. A total number of 598 colonies from 52 soil samples were screened based on their sizes of clear zones and the isolate Aspergillus sp. 558 was selected as a candidate due to its most prominent xylanase activity. In artificial mutation by ultraviolet and ethyl methane sulphonate, 126 colonies derived from the candidate isolate exhibited larger clear zones, indicating enhanced xylanase activity. Of those, the mutants Y8, Y29 and Y120 had clear zones in diameters of 46.3, 41.1 and 40.6 mm, respectively. These measurements were enhanced by 128.6%, 114.2% and 112.8% compared to those produced by the candidate isolate prior to the mutation. After 10 generations of subcultures, the three mutants remained stable in xylanase activity. The mutant Y8 was consistently best in its xylanase activity, reaching 1097 U/g. This mutant produced xylanase, P-glucanase, cellulase and acidophilic protease of 167.7%, 173.2%, 131.0% and 102.5% more than the mother candidate isolate, respectively. The mutant Y8 was identified as Aspergillus awamori Nakazawa by the Institute of Microbiology of Chinese Academy of Sciences.Optimization of medium components and fermentation conditions. Carbohydrate sources were found significantly affecting the production of xylanase by the mutant A.awamori Y8 with wheat or rice bran as a complex carbohydrate source being superior to singular carbohydrate sources such as starch, sucrose, lactose or glucose. Among nitrogen sources influential on xylanase production, yeast extract was better than L-asparagine, soybean meal and NH4NO3 whereas urea, fish meal, (NH^SC^, peptone, KNO2 and KNO3 were poor. The minerals MnSO4 and FeSCU to some degree increased xylanase production but CaCh, Fe2(SO4)3 and ZnSC>4 had insignificant effect. However, MgSC>4 and CuSO4 depressed significantly the xylanase production. With the effects of different compoents on the xylanase production and their market prices in consideration, the medium used for the production of xylanase by A. awamori Y8 was composed of wheat bran as carbohydrate source, soybean meal and NH4NO3 as nitrogen source and MnSO4 as inorganic additive. Based on orthogonal experiments, the medium components were optimized as 37.79% wheat bran, 9.10% soybean meal, 1.51% NH4NO3, and 1.60% MnSO4 plus 50% H2O.For the production of xylanase by A. awamori Y8, an appropriate temperature ranged from 28 to 32°C and the optimum was at 30°C. The initial pH was appropriate at 4.0-6.0 but best at pH 5.5. An inoculum proportion was optimized as 2.0% despite an acceptable range of 1.5-2.5%. A desirable fermentation period ranged 48 to 54 h and the optimum was 50 h. The optimized medium with initial pH 5.5 was inoculated at 2.0% (v/w) of the suspension of fresh spores (107 CFU/mL). After incubation at 30°C for 40-54 h, the activities of xylanase, p-glucanase, cellulase and acidophilic protease in the cultures reached 1126, 422, 39 and 96 U/g, respectively. Thus, the mutant strain A. awamori Y8 was capable of utilizing wheat bran and soybean meal as inexpensive substrate for efficient production of xylanase, P-glucanase, cellulase and acidophilic protease, which are all active components of feed-purpose enzyme preparations against several anti-nutrition ingredients in common feedstuff. This indicates the high potential of A. awamori Y8 as a candidate strain for production of enzyme preparation as feed additive.Purification and characterization of A. awamori Y8 xylanase. After A. awamori Y8 cultures were salted out with (NH^SC^, 30-60% of the crude preparation consisted of 83.9% xylanase. The xylanase-purified ratio was further enhanced to 1.63 and 11.81 throught ion exchange chromatography based on DEAE-Sephadex A50 and DEAE-Sepharose CL-6B, respectively. The singular protein band appeared in SDS-PAGE electrophoresis, indicating a molecular weight of 21.6 kDa for the purified xylanase with an isoelectronic point (pi) of 4.9. When using birchwood xylan as an action substrate, the xylanase activity fit to the kinetic equation v = 2.434[S]/(8.217+[S]). The activity of A. awamori Y8 xylanase was significantlyinhibited by inclusion of Cu2+ in reaction, less affected by Ca2+, Fe3+, Zn2+ and Mg2+, but significantly enhanced by Mn2+ and Fe +. The relative xylanase activities on the substrates of birchwood xylan, larchwood xylan, oat spelt xylan and sodium carboxymethylcellulose were 100%, 78.0%, 105.1% and 0%, respectively. This reveals that the xylanase produced by A. awamori Y8 was a specific enzyme.The appropriate conditions for the action of A. awamori Y8 xylanase were 39-45°C and pH 3.8-4.6, with optima at 42°C and pH 4.2. For the action of the P-glucanase in solid preparation, the ranges of 42-48°C and pH 4.4-5.2 were optional but the optimal regime consisted of 45°C and pH 4.8. Since temperature in alimentary canals of domestic animals and birds are usually around 40°C with a pH range of 2-7 after ingestion, both xylanase and P-glucanase produced by A. awamori Y8 were quite well in accordance with the alimentary environments and thus highly potential for practical application.The activity of pure xylanase from A. awamori Y8 was stable at 40°C for 3 h but this stability was challenged as the temperature increased. For instance, the xylanase activity lost 6.7% at 50°C in 2 h, 31.1 % at 60°C in 1.5 h, and up to 25.2% and 94.4% at 70°C in 10 and 40 min, respectively. Based on the thermal stability assays of a solid formulation, the relative activities of A. awamori Y8 xylanase and P-glucanase were 94.3% and 91.2% for 10-min maintenance at 90°C but decreased to 75.1% and 66.8% in 1 h. This indicates that the solid formulation was temporarily tolerable to the high temperatures of 75-93°C during the granulation of feed manufacturing. Stored under dry conditions at ambient temperatures, the xylanase activity in the formulation remained >95.7% for 7 months while the p-glucanase activity kept >95.9% for 6 months. However, both enzymes kept 77.4% and 67.7% of their activities at the end of 12-month storage, respectively.Pilot production trials and application of xylanase-based preparation. The xylanase-based preparation of A. awamori Y8 was produced in pilot trials using two technological procedures. A multilayer solid fermentation in workshop resulted in high yields of crude enzyme preparations which had xylanase activities close to those under laboratory conditions. This technology was simple and easy at the cost of 3360.8 yuan per ton of crude enzyme preparation. However, it took large working area and was difficult for temperature and humidity control during fermentation and was labor consuming. Another technology tested was solid fermentation in fermentors semi-automatically controlled. With this technology, substrates were sterilized in fermentors and then inoculated by automatically mixing. The temperature and humidity were readily controlled. As a result, working area was saved by onethird and labor saved by a half. The period of fermentation was shortened by one fourth. However, the xylanase activities in the resultant crude preparations on average were only equivalent to 83% in those produced by the multilayer solid fermentation in workshop. This reduction perhaps was attributed to limited ventilation during the fermentation in fermentors.Early indica rice diet including 0.1% A. awamori Y8 xylanase-based preparation was supplied for weaning piglets, growing pigs and finishing pigs for further experiments. The resultant weight gains in the preparation-inclusive treatments increased by 11.33%, 10.86% and 10.47% for the three groups of pigs compared to blank controls. Correspondingly, feed-gain ratios were increased by 5.84%, 4.78% and 5.33% whereas the costs of feed per ton were reduced by 24.22, 52.30 and 48.12 yuan. Moreover, diarrhea percentage of the weaning piglets fed with preparation-inclusive feed was 2.01% lower than that in the blank control group and the degree of their diarrhea was obviously slight. The effectiveness of early indica rice diet added with 0.1% A. awamori Y8 xylanase-based preparation was superior to that of maize-soybean diet. Thus, 0.1% A. awamori Y8 xylanase-based preparation as feed additive was confirmed to improve production of weaning piglets, growing pigs and finishing pigs, to increase daily gain and feed transformation efficiency, and to reduce production cost.Wheat as basic daily food including the enzymatic preparation at the levels of 0.05% and 0.10% xylanase-based preparation was supplied for AA broilers. The resultant weight gains were 7.25% and 7.98% greater than those in blank controls; the feed-gain ratios increased 5-6% and feed cost decreased by 4.67% at the lower xylanase level and 5.67% at the higher level. When feeding Loman laying hens with maize-soybean diet including 0.10% xylanase-based preparation, the feed-egg ratio was decreased by 20.39% compared to blank controls but laying rate was increased by 19.40%.