| Palm kernel cake is a residual products of palm oil processing,and the proportion of nutrients like crude protein,crude fat and crude fiber is moderate,so it possesses elevated feeding value.mannan is one of the primary components,accounting for about 35% of dry weigh.Animal organism beyond digest mannan in absence of relevant enzyme system,therefore,mannan is considered as a kind of antinutritional factor,which limits the absorption and utilization of nutrients in feed.β-mannase is a sort of hemicellulase which can catalyze β-1,4 glycosidic bonds,and effectively eliminate various antinutritional factors in feed,boosting animals to digestion and absorption of nutrients,and improve the utilization rate of palm kernel cake fermented feed.As a functional prebiotic,the enzymatic hydrolysis product mannan oligosaccharide also populate digestive track with beneficial microbes.The development of β-mannase was established a green,environmentally and economical channel make feed materials such as palm kernel cake bring into play application value.There are some matter of β-mannase deficient in catalytic activity and stability in the process of agricultural breeding.β-mannase is liable to forfeit biological activity in the high temperature environment of feed processing and the acidic environment of animal gastrointestinal tract,bring about the application effect is awful.In this paper a strain which can efficiently degraded mannan into oligooligosaccharide was preserved in the laboratory as the research object,the characteristic of β-mannase were studied combined with genetic engineering and molecular biology technology,furthermore,we modified its enzymatic properties based on computer-aided design and obtained β-mannase with stability and efficient catalytic activity under high temperature and acidic conditions eventually,response surface methodology was used to optimize the technical system of β-mannase degraded antinutritional factors in palm kemel cake,The results of the study are showed below:（1）In this study,a strain which can efficiently degrade mannan was preserved in the laboratory as the research object,the result of 16 Sr DNA identification showed the strain belongs to Bacillus subtilis.The gene of encoding β-mannase was obtained by PCR amplification and named manDL4,it belongs to the glycoside hydrolase 26 family.Escherichia coli BL21 was used as heterologous expression host,the enzymatic characteristic were studied,results showed that the enzyme weight was41.5 k Da,optimal p H and temperature were 6.5 and 60.0 °C,respectively,after tolerated for 60 min between p H 5.5 and 10.0 the relative residual enzyme activity was maintained more than 80%,after incubated at 60 ℃ and 65 ℃ for 100 min the remaied enzyme activity was above 40%.（2）Eight potential disulfide bridges were designed with the Disulfide Design 2.0,and the mutants of thermal stability were improved finally by constructed mutants expression vectors,the half live of manDL4 was 40 min at 65 ℃,N304/E332-C,H146/T149-C,T106/D111-C was 100 min,100 min,80 min respectively,2.5 folds,2.5 folds and 2.0 folds of wild type,A303/E332-C remained 67.4% of enzyme activity after tolerated 100 min,but the mutant H146/F142-C showed a significant decreased in enzyme activity after tolerated 60 min,only 13.5% of relative enzyme activity remained.The half lives of A303/E332-C,N304/E332-C,and H146/T149-C,were nearly 100 min,80 min,and 55 min respectively at 70 ℃,which was 5.0 folds,4.0 folds,and 2.75 folds of wild type,respectively.However,the temperature tolerance of the mutants H146/F142-C,H146/N139-C,E105/G145-C,T106/D111-C,and I151/ Q155-C was decreased.（3）Introduced mutation at conservative sites by alignment of multiple sequence and optimization of surface charge,the result showed the enzyme activity of mutants K322 E,Q143R/K322 E,and L242 I was increased by 29.6%,25.9%,and15.6% compared with wild type respectively,the relative enzyme activity of mutant K322 E remained above 90% after toleranced between p H 4.0 and 12.0 for 60 min,but the temperature stability of all mutants was decreased with different degrees.V337 E,K319E,H346 D,Q90E,K339 E,N186D can still retain more than70% of the relative enzyme activity in buffer solution with p H 4.0 for 60 min,while the wild type only retain 55% of the relative enzyme activity.（4）In this paper,the antinutritional factors of palm kemel cake digested with manDL4 were analyzed under the following conditions: time,p H,temperature,substrate content,and amount of enzyme,the time,p H,and temperature play a significant effect on reducing sugar content.Combined with response surface analysis the optimal conditions of enzymatic palm kemel cake by manDL4 were determined,when the enzymatic hydrolysis time,p H and temperature were 7.46 h,5.46,55.69 ℃respectively,the highest reducing sugar content produced by enzymatic palm kemel cake was 7.43 mg/m L,the best scheme of enzymatic hydrolysis of palm kemel cake was obtained,which provided some experiment reference for enzyme hydrolyzed anti-nutritional factors in palm kemel cake.In summary,we focused on a selected strain that can degraded mannan with high efficiency,The enzymatic characteristic of manDL4 was studied with the assist of genetic engineering and molecular biology technology,we introduced disulfide bonds through the design of Disulfide Design 2.0 to improved thermal stability and the enzyme activity of manDL4 with rational design,ultimately,the technological system for enzyme hydrolyzed hydrolysis of antinutritional factor of palm kernel cake by manDL4 was optimized by combinated with response surface. |
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