Effect Of Co-Expressing Adjuvant Prptein On Mannanase And Protease Production In Pichia Pastoris

?-mannanase and protease are important industrial enzymes,which play an important role in food,medicine,feed and other fields.The effects of co-expressing auxiliary proteins on mannanase and protease of Pichia pastoris were studied by co-expressing VHb and Hac1p molecular chaperone protein in Pichia pastoris expression system.The main findings are as follows:(1)According to VHb's accession number(AY278220)in Gen Bank,the original sequence of VHb was found.The codon optimization of vgb was carried out,and then the optimized vgb was synthesized.Primers were designed to amplify vgb in vitro by PCR,and p PIC9K-vgb expression vector was constructed.The mannanase engineering strain Z?A-man-GS115 and the protease engineering strain Z?A-pk-GS115 were electroporated.The results showed that the enzyme activity of the strain without vgb was significantly lower than that of the strain with vgb under oxygen restriction condition.The mannanase activity of strain man-vgb-GS115 10#was the highest,reached 2452.43U/m L,which was 3.85 times of contrast group;the strain of pk-vgb-GS115 6#produced the highest protease,reached 1369.41U/m L,which was 2 times of contrast group.However,there is no obvious difference under the condition of unlimited oxygen.(2)According to the gene sequence number(8196642)of hac1 in Gen Bank,primers were designed to amplify hac1 gene in vitro with GS115 genome as template,and p PIC9K-hac1 vector was constructed.The vector was electroporated into Z?A-man-GS115 and Z?A-pk-GS115 respectively.The results showed that there was no significant difference between hac1 and non-hac1 strains in enzyme activity.(3)Optimum temperature and methanol tolerance test results:the selected man-vgb-GS115 10#,pk-vgb-GS115 6#,man-hac1-GS115 9#,pk-hac1-GS115 10#were carried out separately 1%,2%,3%methanol tolerance experiments,the results showed that the methanol tolerance of man-vgb-GS115 10#and pk-vgb-GS115 6#was significantly higher than that of the contrast group,indicating that the methanol tolerance of engineered strains integrated with vgb has been significantly improved,while man-hac1-GS115 9#and pk-hac1-GS115 10#did not show good methanol tolerance;setting different fermentation temperatures(20?,25?,30?),man-vgb-GS115 10#,pk-vgb-GS115 6#have better fermentation effect at 30?,while man-hac1-GS115 9#,pk-hac1-GS115 10#have better fermentation effect at20?,indicating that it is more conductive to its expression under low temperature conditions.(4)Enzymatic properties of mannanase:the optimal reaction temperature and p H were 55?and 5.5 respectively;Ca²⁺,Mg²⁺,Mn²⁺promoted the activity of mannanase;temperature and p H stability tests showed that the mannanase was most stable at 45?and p H 5.5 respectively;the mannanase was stable to Tween 80,glycerol,acetone,ethanol,dimethyl sulfoxide and so on.(5)50L fermentation tank magnification test result:the strain man-vgb-GS11510#integrated with the vgb gene under the condition that the dissolved oxygen is controlled below 10%±5%,the mannanase activity is 2.4 times that of the contrast group,reached 45679.47U/m L;while the strain man-hac1-GS115 9#integrated with the hac1 gene increased by 14.29%to 40259.25U/m L under normal dissolved oxygen;the strain pk-vgb-GS115 6#under the condition of controlling dissolved oxygen at 10%±5%,the enzyme activity of protease was 2.3 times that of contrast group,reached 28743.89U/m L;the strain pk-hac1-GS115 10#integrated with hac1gene was under normal dissolved oxygen conditions,the enzyme activity increased by 7.4%,reached 17893.54 U/m L.