Expression,directed Evolution And Bread Baking Application Of Maltogenic Amylase From Bacillus Licheniformis R-53

Maltogenic amylase(EC 3.2.1.133)has unique catalytic versatility and multi-substrate characteristics.It exhibits unique activity to hydrolyze both?-D-(1,4)-and?-D-(1,6)-glucosidic,quickly utilizes amylose and amylopectin as substrate,and is beneficial to extend the shelf life of bakery products.Therefore,maltogenic amylase has application advantages in the bakery industry.In this study,a strain of Bacillus licheniformis with high yield of maltogenic amylase was selected from soil samples,and the maltogenic amylase BLMA gene was cloned from the genomic DNA of the strain.The food-safe strain B.subtilis WB600(GRAS product approved by the U.S.Food and Drug Administration)was used as host cell to construct a heterologous expression system B.subtilis/p MA0911-BLMA.And the properties of recombinant enzymes were studied.The recombinant maltogenic amylase BLMA was applied to dough making and bread baking,and the positive effects of BLMA on improving the rheology of dough and the quality of bread during storage were investigated.By the directed evolution technology,a mutant with improved enzyme activity,the higher optimum temperature and thermostability was obtained to futher improve the effect of maltogenic amylase BLMA in bread baking.This research provides a new type of maltogenic amylase with good effects for the bread baking industry,which is conducive to promoting the development of the industry.The main results are as follows:(1)A strain of Bacillus licheniformis R-53 with high maltogenic amylase production was obtained.Then the maltogenic amylase BLMA gene was cloned from the genome of B.licheniformis R-53 using chromosome walking technology.The gene fragment of maltogenic amylase BLMA is 1707 bp in length,encoding 568 amino acids.The phylogenetic tree and amino acid sequence conserved domain analysis showed that the gene encodes a novel maltogenic amylase.(2)The recombinant expression strain B.subtilis/p MA0911-BLMA was constructed,and the properties of the recombinant maltogenic amylase were determined.The recombinant maltogenic amylase BLMA exhibited the highest enzyme activity of 3235.26 U·mg^-1,its optimum temperature was 60°C and the optimum p H was 6.5;after 30 min of treatment at80°C,the residual enzyme activity could be maintained 60%.Mn²⁺?Ba²⁺?Cu²⁺?Fe²⁺?Zn²⁺?EDTA and SDS could inhibit the enzyme activity,while Ca²⁺could increase the activity of BLMA by 1.36 times,indicating the recombinant enzyme was Ca²⁺-dependence.(3)The Mixolab was used to measure the properties of the dough,it was found that adding maltogenic amylase BLMA at optimal concentration(60 mg·kg^-1)was beneficial to reduce the retrogradation of starch and significantly improved the rheological properties of the dough.Three different maltogenic amylases(Novamyl 3D BG,MAM100 and BLMA)were added to study the effects of BLMA on the quality of baking bread.The bread with BLMA had a 32%higher specific volume than bread without enzyme;the elasticity and sensory of this bread during storage were significantly improved;and the hardness was reduced by 2.12 times after storage for 10 days.When compared with the commercial enzyme MAM100 and Novamyl 3D BG,BLMA showed better effect on improving the specific volume of bread,and had similar effects to Novamyl 3D BG in terms of reducing hardness,improving elasticity and maintaining sensory.The results indicated that BLMA could significantly inhibit the aging of bread and prolong the shelf life of bread.(4)Random mutations of gene were carried using error-prone PCR technology.The effective mutation sites were determining by sequencing mutant stains F1 and F2 with improved enzyme activity and thermostability,and after single-site and double-sites directed mutation,a mutant BLMA1 with improved enzyme activity,optimum temperature and thermostability was obtained.Compared with wild-type BLMA,the enzyme activity of mutant was as high as 2.16times,and the optimum temperature was raised to 65°C;after treatment at 55-80°C for 30 min,the residual enzyme activity was above 60%,suggesting its good thermostability.The results of dough rheology showed the effect of 45 mg·kg^-1 mutant was similar to 60 mg·kg^-1 wild-type BLMA.During storage,the effect of 45 mg·kg^-1 mutant on maintaining bread elasticity and reducing bread hardness was better than 60 mg·kg^-1 BLMA,the mutant was more conducive to extending the shelf life of bread.