Study On Protease Gene Shuffling And Expresssion And Its Application In Feather Meal Enzymatic Hydrolysis And Broiler Production

With the shortage and rising price of feed protein resources, how to improve the quality of protein resources such as feather meal and blood meal has become more important than ever before. Although the feather meal is rich in protein, it is difficult to be hydrolyzed by normal protease and digested by animal due to its stable and complicated structure.In this study, the neutral and alkaline protease genes from Aspergillus oryzae and Bacillus subtilis which can degrade feather meal effectively was isolated and cloned. After two cycles of DNA shuffling, an ideal protease gene named NPGA-S2 was selected by the sizes of transparent zones around colonies on casein plates. It was then transformed into Pichia pastoris, and the protease activity and character of recombinant Pichia pastoris was determined. Study on fermented feather meal of recombinant P. pastoris and its effect on production performance of yellow-feather broilers was conducted. The main results were as following.(1) Two strains of Aspergillus oryzae and Bacillus subtilis were selected from five microbial strains by determining protease activity. The biodegradability of feather meal by the two strains was measured by the contents of soluble protein and protease activity in the fermented feather meal. After 72 h fermentation of Aspergillus oryzae and Bacillus subtilis, the protease activity in feather meal was raised to 3256.61 U/g(P<0.05) and 690.03 U/g(P<0.05), respectively. The soluble protein contents of feather meal fermented by Aspergillus oryzae was increased from 2.21 mg/g to 362.07 mg/g(P<0.05) after 48 h incubation.(2) Four kinds of protease genes(neutral and alkaline protease genes from Aspergillus oryzae, neutral and alkaline protease genes from Bacillus subtilis) were successfully amplified using RT-PCR technique. Compared with the published gene sequences in NCBI, four protease genes had 99.5% homology at least.(3) After DNA shuffling of protease genes, the purified PCR products were inserted into p MD19-T vector and transformed into E.coli competent cells by heat shock method. Three mutant neutral protease genes of Aspergillus oryzae named as NPGA-S1, NPGA-S2, NPGA-S3, and one mutant neutral protease gene of Bacillus subtilis named as NPGB-S1 were obtained with casein plates.(4) Four mutant protease genes and two native neutral protease genes were transformed into competent P. pastoris with electroporation. NPGA-S2 and NPGA-S3 mutants were found to express the maximal protease activity in the recombined P. pastoris.(5) The second cycle of DNA shuffling was carried with NPGA-S2 gene, NPGA-S3 gene and four native protease genes. A new mutant protease gene of Aspergillus oryzae named NPGA-S4 was selected and transformed into Pichia pastoris. Compared with the other recombined P. pastoris strains, the maximal protease activity was detected in the recombined P. pastoris containing NPGA-S2 gene(P<0.05). Sequence analysis showed that the base in 1022 th position of NPGA-S2 protease gene was changed from thymine to cytosine, inferring that cysteine was changed to arginine in the mutant protease. After 48 h incubation for the transformed P. pastoris with the NPGA-S2 or native protease genes, the NPGA-S2 protease activity was 36.4% higher than the native protease(P<0.05). The optimal p H and temperature of the mutant protease were 6.5-8.0 and 30-70°C, respectively, which indicated better stability than the native protease(P<0.05).(6) Feather meal was fermented by Aspergillus oryzae and recombined P. pastoris containing NPGA-S2 or native genes. It was found that the nutrients apparent metabolic rate and the amino acid contents of the fermented feather meal were higher than the control,especially lysine content was increased about 1 fold than the unfermented feather meal.(7) In order to study the effect of replacing fish meal with fermented feather meal on production performance of yellow-feather broilers, growth performance, slaughter performance, meat quality, intestinal microbe, blood biochemical index were determined. At the same time, economic benefit was also analyzed.Feeding trial was divided into two phases, earlier(1-28 d) stage and later(29-56 d) stage. A total of 250 one-day-old yellow-feather broilers were assigned to 5 groups, 50 broilers for each group consisting of 5 replicates. Group A(Control group) was given with basal diet containing fish meal; group B was fed with feather meal fermented by Aspergillus oryzae replacing the same percentage of fish meal in the basal diet; group C(Negative control group) was fed with feather meal fermented by recombinant Pichia pastoris without protease gene replacing the same percentage of fish meal in the basal diet; group D was fed with feather meal fermented by recombinant Pichia pastoris with native protease gene replacing the same percentage of fish meal in the basal diet; group E was fed with feather meal fermented by recombinant Pichia pastoris with mutant protease gene(NPGA-S2) replacing the same percentage of fish meal in the basal diet. The nutrient values of four groups were balanced with the control group.The result showed that the average daily gain(ADG), average daily feed intake(ADFI), feed conversation rate(FCR) and feed nutrients metabolic rates in two phases were not significantly different among four groups(P>0.05) except for group C(P<0.05). The carcass recovery, eviscerated rate and meat quality were not significantly different among five groups(P>0.05). The protease activities of intestine in group B, group D and group E were higher than that in group A and group C(P<0.05). The intestinal bacterium populations, similarity coefficient and diversity were roughly the same among five groups. Except for group C, the serum total protein, globulin and albumin were not significantly different in the other groups. The serum enzyme activities and immune system were not be affected by the fermented feather meal replacing fish meal in basal diet. The better economic benefits were also obtained by replacing fish meal with the fermented feather meal in basal diet for broilers, which indicated that this technique would be useful for improving the unconventional protein feedstuffs.