Study Of Heterologous Expression And Enzymatic Properties Of Oligo-1,6-glucosidases From Different Bacteria

In the industrial lactic acid fermentation by Bacillus coagulans using corn flour/starch hydrolysate as substrate,the high residual sugar at the end of fermentation not only reduces the conversion ratio from glucose to lactic acid,but also increases the cost for lactic acid separation and purification,decreases the product quality and shortens their shelf life.Isomaltose accounts for about 60%of the total residual sugar in lactic acid fermentation broth.Oligo-1,6glucosidase（EC 3.2.1.10）showed activity for the utilization of isomaltose.In this paper,based on the previous research work and approved by BRENDA database search,five possible heatresistant and acid-resistant oligo-1,6-glucosidase enzyme genes（Bacillus coagulans ATCC 7050:malL BF29₂011（BC1）and malL BF29₂004（BC2）;Bacillus subtilis 168:yvdL BSU34560（BS1）and yugT BSU31290（BS2）;Bacillus thermoglucosidasius KP 1006:malL（KP））were selected for the research of recombinant expression in E.coli,purification and enzymatic properties of these oligo-1,6-glucosidases,which would lay the foundation of genetic engineering of Bacillus coagulans and screening of efficient fusion strains for lactic acid fermentation.First of all,based on the feasibility of fermentation culture,the ability of B.coagulans ATCC 7050 and B.subtilis 168 to utilize glucose and isomaltose were studied.It was found that both strains could use glucose and isomaltose for growth when cultured with glucose or isomaltose as only carbon source,and Bacillus coagulans ATCC 7050 had higher substrate specific consumption rate and faster growth rate than B.subtilis 168 in the early fermentation period with either glucose or isomaltose.In the fermentation with mixed carbon source of glucose+isomaltose,both strains peferred using glucose for growth first,and then started to use isomaltose for secondary growth after a certain stagnation period after glucose depletion,which showed obvious catabolite repression.In addition,B.coagulans ATCC 7050 showed stronger ability to use isomaltose once again.Secondly,the plasmid pET-22b（+）was used as an expression vector to construct oligo1,6-glucosidase gene expression system.Five oligo-1,6-glucosidases were successfully expressed in E.coli BL21（DE3）and purified by nickel column affinity chromatography.Finally,the enzymatic properties of the above five oligo-1,6-glucosidases were studied.They all belonged to acidic enzymes,and the optimal pH of BC1,BC2 and KP was 5.0,and that of BS1 and BS2 was 6.0.The optimal temperature of these five oligo-1,6-glucosidases from high to low was KP（60℃）>BC1（56℃）=BC2（56℃）>BS1（43℃）＝BS2（43℃）.In addition,Mg2+and NH4+ could enhance the enzyme activity of BC1,while Co2+,K+,NH4+ could enhance the enzyme activity of KP.However,these ions had no obvious activating effect on BC2,BS1 and BS2.Zn2+,Cu2+and Ni+ all had obvious inhibitory effects on these five oligo-1,6glucosidases.When p-nitrobenzene-α-D-glucoside（pNPG）was used as the substrate,the conversion number and catalytic efficiency of these enzymes from high to low were KP>BC1>BS1>BS2>BC2.The substrate utilization characteristics of five oligo-1,6-glucosidases were studied by using maltose,isomaltose,sucrose,lactose,trehalose and soluble amylose considering the components of residual sugar in lactic acid fermentation broth.The most suitable substrate for BC1,BC2,KP and BS1 was isomaltose,among which the catalytic activity on isomaltose of BC1 and KP reached 12.82 mmol·min-1·g-1 and 7.24 mmol·min-1·g-1,respectively,and the most suitable substrate for BS2 was maltose,its catalytic activity on maltose reached 4.11 mmol·min-1·g-1.