Research On A-cyclodextrin Glycosyltransferase High-yield Strain Breeding And Enzyme Production Conditions

Cyclodextrin （referred to as CDs） is the general term for a series of cyclicoligosaccharides, which are the product of enzyme catalysis. Cyclodextringlycosyltransferase （abbreviated as CGTases, EC2.4.1.19） produced by certainmicroorganism species can transform its substrates （the polysaccharide） intoCyclodextrin. Their naming principle is based on the number of ring-formingglucose group: cyclodextrin, consisting of6glucopyranosyl units known asα-cyclodextrin, and β-cyclodextrin（7rings）,γ-cyclodextrin（8rings）.The CD molecule is shaped as a hollow tube with wide upper part andnarrow lower part, and two open ends. Its rim is hydrophilic and its internalcavity is hydrophobic, while all the hydroxyl groups are in the outside of themolecule. The primary hydroxyl of cyclodextrin forms a small tapered mouth,and its secondary hydroxyl forms a large tapered mouth.The special molecular structure of CD can envelope the different compounds,such as organic molecules, rare gases, inorganic compound, etc. and change theirphysical or chemical properties substantially. CD has a very high research valuein the field of chiral separation, molecular recognition, mimic enzyme, newmaterials and super-molecular chemistry. CD has got great attentions and beenwidely used in food flavors, fragrances, analytical detection, agricultural breeding,cosmetics, moisturizer, chemical industry, environment protection and otherfields.CD production is a systematic project. In present days, CD is mainlyproduced by enzymatic method. Under certain conditions, CGTase acts onstarch, glycogen and malt oligosaccharide and generates CD through catalysis,cyclization, coupling and disproportionation reactions. The raw materials ofproducting CD are including corn starch, tapioca starch, potato starch andmaltodextrin. Studies have shown that a variety of Bacillus strains can produce CGTases. They are Bacillus macemns, Bacillus megaterium, Bacillus circulans,Bacillus stearothermophilus, Brevibacillus brevis, Bacillus alcalophilus andKlebsiella oxytoca. Most CGTases can convert starch or its hydrolyzate intoα-CD, β-CD and γ-CD. However, the proportion of the three CD forms variesaccording to different CGTases obtained from different microorganism strains.A-CGTase is an enzyme essential for the production of α-CD. In order toimprove the yield of α-CD, it is crucial to breed a high-yield strain of α-CGTase.In this study,22strains from starchy soil samples which produced α-cyclodextringlycosyltransferase were isolated. Among them three strains which had highenzyme activity were screened out with the circle method by comparing the sizesof transparent ring. Finally, by plotting the enzyme activity curve, a strain withthe enzyme activity of0.64U/mL was obtained and called N1. With its colonymorphology characteristics and physiological and biochemical tests, the N1wasidentified to be acid-producing Klebsiella Enterobacteriaceae.In order to further improve the level of α-CGTase enzyme production,mutagenesis methods such as UV, nitrite, DES, and nitrite+DES were used.Combined with tablet rapid screening a higher production α-CGTase strain D6was obtained. Its enzyme activity was5.68U/mL, increased by787%comparedwith the original strain N1. After continuous passage culture for eight times andthe enzyme activity was determined, the enzyme activity of mutant D6wasbasically stable. The D6was chosen as the α-CGTase high-yield strain.Fermentation conditions for enzyme production are also crucial. Thefermentation conditions of α-CGTase were also studied. The conditions includedmedium composition and culture conditions. With single factor and orthogonalexperiments to determine the optimum fermentation conditions, they were: potatostarch10g/L,8g/L peptone,4mmol/L MgSO₄, initial pH7.0, culture temperature40°C, inoculums size8%with volume of20ml.