| Neutral protease is one of the earliest protease preparations for human founding,and they are widely used in food?medical?leather and washing industry.Neutral protease is a kind of protease for cut inside,the optimum pH was between 6.0 to 7.5,and it has a series of action for pure natural?safe non-toxic?strong hydrolysis ability and so on.It also can hydrolyze the protein macromolecule into short peptide and amino acid.Proteases for the application of the market at present are mostly coming from bacteria.The quality of neutral protease product can reach the standard of food grade and pharmaceutical grade.With the adoption of biological engineering technology,microfiltration,ultrafiltration membrane separation and vacuum freeze drying technology.Aspergillus oryzae,a fungus widely used in the production of traditional Asian foods such as rice wine,soy sauce,and soybean paste,secretes abundant hydrolysis enzymes such as amylases and protease.Oryzae is considered to be a safe organism to produce proteases used in the food industry.Oryzae proteases show high efficiency in the hydrolysis of plant protein,such as soybeans.It was recently reported that a crude protease extract from Oryzae produced a higher degree of peanut meal protein hydrolysis,compared to commercial proteases Alcalase,Protamex,and papain.In addition,its hydrolysate had better sensory taste than other Hydrolysates.To acquire higher production of oryzae proteases for characteristic identification and utility,gene engineering technology has been used to overexpress these recombinant proteins.Among the A.oryzae proteases,two neutral metalloproteases,neutral protease I(NpI)and neutral portease II(NpII),both of them belong to the zinc-dependent class of metalloendopeptidases.This study aimed to obtain high-level expression Pichia pastoris GS115 strains secretaryly expressing recombinant neutral protease I from Aspergillus oryzae 3.042 for industrial purpose.The modified coding sequence of the target gene was modified and synthesized,followed by cloning into the expression vector PHBM905BDM,which harbor d1+2×201 AOXl promoter and MF4I leader sequence.And then shifted to Pichia pastoris GS115 with the electricity,Casein plate method could screen out high expression of the neutral protease.Preparing neutral protease I within a 5 L fermentor can reach high level.The yield of target protein reached 12.87 mg/ml and the enzyme activity was about 49000 U/ml,so neutral protease I can be applied to industrialization production.It was shown that the enzyme activity of the recombinant neutral protease in triangle flash reached 1266.64 U/ml,and the protein concentration was 1.017mg/ml.Enzymatic property of the recombinant neutral protease I are as follows,the optimum temperature and pH were 55? and 8.0,respectively.Consistent with previous reports,NpI had high activity in a broad temperature and pH range.The optimum pH of this enzyme was pH7.0-9.0.About 80%of its activity was retained in this range.The optimum temperature of NpI was 45-55oC and above 80%of activity was remained.This enzyme was extremely sensitive to EDTA,which is consistent with previous report that this enzyme is a protein which is zinc-dependent.Our result indicated that low concentration of zinc stimulated the activity while high concentration inhibited its activity.EDTA inhibited almost all of its activity,and Cu2+?Fe2+ and Co2+ inhibited much.Mn2+ inhibited slightly of its activity.On the other hand,Ca2+?Mg2+ and PMSF stimulated its activity.Meanwhile,the results of our study indicated that the specific activity this recombinant enzyme was 3600 U/mg and 2600 U/mg after purification.This decrease may due to the remove of ions such as calcium and magnesium.The amino acid sequence of the neutral protease I with a variety of physical and chemical properties are similar to the thermolysin,they both belong to the thermolysin family. |
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