Characterization And Application To Pig Feedstuff Of The Enzymes From Aspergillus Niger

Because Aspergillus niger (An) could produce many kinds of hydrolatic enzymesoutside the cells, we obtained An SL2-111 which could produce complex enzymes highlyfrom An FJ-008, and optimized the fermentation process. Characterization, amino acidcomposition, kinetic property and chemical modification of acid protease from An SL2-111were also discussed in this dissertation by using leaching and purification. At the meantime,the gene of acid protease also was cloned and sequenced by RT-PCR and PCR. Moreover,128 weaned pigs (Landrace×Yorkshire, 5 weeks old) were used in a 9-week experiment andassigned into 4 treaments to evaluate complex enzymatic preparation from An SL2-111 ongrowth performance, apparent digestibility and blood biochemical index in pigs.1 About 1260 strains were gained from An FJ-008 by treating with UV as well ascomplex mutagen of UV and NTG, and among them, there were 139 strains which mutateddifferently. As a result, we selected 16 strains in accordance to the size of hydrolysis circle.In addition, correlation analysis by SPSS10.0 showed that there was positive correlationbetween the activity of acid protease and pectinase, acid protease and cellulase. Accordingto this result, the activity of acid protease could be regarded as the response in selecting thestrains. And then, An SL2-111 which produced acid protease highly was screened byexperiment.There was significant difference between An SL2-111 and An FJ-008. The enzymeactivities produced by An SL2-111 were 4525 U/g for acid protease, 7015 U/g for pectinaseand 4497 U/g for cellulase, which were enhanced 97.9%, 70.1% and 13.4% compared to tAn FJ-008, respectively. Its ability of producing enzymes remained steadily after 5 times ofsubculture.2 The solid fermentation condition of An SL2-111 was studied by using single-factorresearch and orthogonal test basing on the response value of acid protease. The resultsshowed that the medium with high content of carbon and low content of nitrogen was fit forthe growing of An SL2-111. The fermentation process could be divided into two phases,one was growing phrase, the other was spore and enzyme producing phrase. In the formerphase, there was a lot of mycelia and hardly enzyme produced. The activities of acidprotease, pectinase and cellulase were 6428 U/g, 8245 U/g and 4911 U/g respectively when An SL2-111 was cultured in the optimal medium and 250 mL Erlenmeyer flask under theoptimal culture conditions.3 The study which investigated the leaching of acid protease from solid fermentationmedium with different solutions showed that 0.1 mol/L lactic acid-sodium lactate buffer(pH 3.5) was the best. Compared with that of 2% NaCl, the recovery ratio was enhanced by50%. Following this, to determine the optimum leaching factors, acid protease was leachedwith various pH, time, solution and leaching ratio. The results indicated that 0.1 mol/LNa₂HPO₄-Citric acid buffer (pH 5.0) gave optimal recovery lasting for 60 min under 40℃and volume ratio of buffer to medium was 10:1. The study of crude acid protease showedthat the optimum pH was 3.0 and the optimum temperature was 55℃. The activity of acidprotease was very stable under condition of pH 3.0～6.0, and acid protease showedfavorable thermal stability under 30℃and 40℃. The results of stability experiment ofcrude acid protease indicated that 5% butanol, 5% potassium sorbate, NaCl, gelatin,xanthan and starch were in favor of conservation. Especially, 1% xanthan could conservecrude acid protease during 45 d under 25℃.4 Acid protease prouduced by An SL2-111 was purified to elecrophoretichomogeneity by the steps of ammonium sulfate precipitation, DEAE-Sepharose Fast Flowcolumn chromatography, Sephacryl S-200 column chromatography and high performanceliquid chromatography. Its purity was checked to be about a single band by SDS-PAGE andthe molecular weight was about 47×10³. The optimum pH was 3.0 and the optimumtemperature was 60℃. The activity of enzyme was very stable under condition of pH 4.0～5.0 and enzyme showed favorable thermal stability under 40℃and 50℃. Cu²⁺ and Mn²⁺showed an obvious activation to the acid protease, on the other hand, Hg²⁺ and Ag⁺ slightlyinhibited the enzyme activity. The acidic amino acid was 17.29%, the alkaline amino acidwas 4.50% and the neutral amino acid was 38.50% ,others was nonpolar amino acid. TheN-terminal partial sequence was SKGSAVTTPQ ,which indicated a high homology withother protease produced by Aspergillus.The difference of hydrolytic ability of bovine haemoglobin, bovine serum albumin, eggalbumin and casein was discovered. The results showed that Km of casein was 0.22 g/L,Vmax of casein was 555.56μg Tyr/min mL, and then Vmax/Km of casein was 2525.27. Ina word, casine was the optimum substrate of acid protease.5 We studied the effect of modification reagents which included NBS, PCMB, N-AI,DTT, Acetyl acetone, MA, BrAe and CDC on the acid protease activity. The results of the chemical modification demonstrated that tryptophan residue, histidine residue, tyrosineresidue and disulfide were essential, while amino-group, sulthydryl-group, carboxyl-groupand arginine residue were not essential for the activity of acid protease.6 Acid protease gene of An SL2-111 was amplified by reversetranscriptase-polymerase chain reaction (RT-PCR) and polymerase chain reaction (PCR).The results indicated that the gene was 1339 bp in length and had 4 exons and 3 introns,which code for a polypeptide of 446 amino acid. The 70-79 of N-terminal partial sequencewas SKGSAVTTPQ by deducing. The sequence of acid protease gene indicated a highhomology with the genomic DNA of aspergillopepsin I from other strains of An andAspergillus saitoi.7 The activities of complex enzymatic preparation produced by An SL2-111 were6290 U/g for acid protease, 4909 U/g for cellulase, 5196 U/g for pectinase and 52385 U/gfor amylase. About 128 weaned pigs (Landrace×Yorkshire, 5 weeks old) were used in a9-week experiment including 3 stages and assigned into 4 treatments to evaluate complexenzymatic preparation on growth performance, apparent digestibility and bloodbiochemical index in pigs. The ration of 4 treatments was added to complex enzymaticpreparation and the levels of addition were 0%,0.5%,1.0% and 1.5%.The results showedthat addition of complex enzymatic preparation reduced feed consumption and increased inweight. The apparent digestibilities of raw fiber, crude protein, crude ash, Ca and P alsowere improved remarkably (P＜0.05), and the contents of total protein, albumen, globulin,glucose and urea nitrogen in serum were increased various degrees. Moreover, theconstruction and function of heart, liver and kidney of pigs were not affected. Among 4treatments, the effect of 1.0% was the best. Growth performance of 3 stages was increasedby 13.57%,30.47% and 34.10% respectively with supplementation of 1.0% of complexenzymatic preparation, and ratio of feed and meat of 3 stages was decreased by 21.26%,9.88% and 13.11% respectively. Futhermore, apparent digestibility of nutrient matter hadsignificant difference as compared with control(P＜0.05).