Study On The Fermentation Conditions Of β-glucanase Producted By Genetically Engineered Strain

β-1,3-1,4-glucanase（EC3.2.1.73, hereinafter referred to as the β-glucanase） is one of themost important enzymes in beer and feed industries, it is also meaningful in researching anddeveloping β-glucan functional food and in human nutrition and health. At present, thedomestic production of β-glucanase is mainly preferred to the mutation strains of Aspergillusand Bacillus, but the amout of enzyme production or thermostability is insufficient ideal. Thisstudy used a genetically engineered strain CA as material, systemically discussed thefermentation conditions of CA, purification, properties and protection of β-glucanase, theβ-glucanase produced by CA is good excretory, high enzyme activity and thermal stability.Based on initial culture medium TB, the producing conditions of β-glucanase wereoverall evaluated by taking biomass and β-glucanase activity as two indexes. The optimalcomposition of culture medium in flask were as follows: sucrose2g/L, yeast extract20g/L,peptone16g/L, KH₂PO₄2.31g/L, K₂HPO₄·3H₂O12.54g/L, CaCl₂0.44g/L, and Tween800.8%（V/V）. The optimal culture conditions obtained by single and orthogonal experimentswere as follows: inoculation volume2%（V/V）, sample loading quantity25/250mL, initial pH6.2. Through these two steps optimization, the activity of β-glucanase could achieve to720.76±11.00U/mL after CA was fermantated in37℃200r/min, improved85%compared withthe initial389.59±17.93U/mL.Based on the aforementioned conditions, IPTG and lactose were chosen as two inducersrespectively, the optimal inducing conditions of different inducers were determined. Theoptimal conditions of IPTG were as follows:1.4mmol/L（terminal concentration） IPTG wasadded after inoculating4h when OD600≈2.5, then the temperature was changed to41℃,and CA was induced for6h; The optimal conditions of lactose were as follows:10mmol/L（terminal concentration） lactose was added after inoculating6h when OD600≈3.8,then the temperature was changed to41℃, and CA was induced for8h. The activity ofβ-glucanase achieved according to these two conditions were2.60and2.52times than theinitial conditions respectively. Although using lactose as inducer prolonged the period offermentation, the effect could achieve to97%of IPTG, and it was safe and thrifty，so it was acertain substitution.5L fermentation tank was used for amplification experiment induced by lactose. Thebiomass and the activity of β-glucanase were3.92±0.05g/L and1221.26±26.22U/mLrespectively in batch fermantation, while they were3.14±0.07g/L and988.74±31.14U/mLrespectively in flask fermantation, they were improved24.8%and23.5%respectively in batchfermentation compared with flask fermentation. It provided data base for large scaleproduction of β-glucanase by CA induced by lactose.The β-glucanase was electrophoretically pure after being purified by Ni2+chelatechromatography. The characteristics of β-glucanase were studied, the optimal reactiontemperature was70℃, the thermal stability was good in40⁶0℃, the optimal reaction pH was7.6, the pH stability was good in6⁸, Ca²⁺and Fe³⁺had certain activating effect for theactivity of β-glucanase, and Na⁺had the strongest restraining effect.The composition of complex protective agents was confirmed by single and orthogonalexperiments: bovine serum protein0.75%（W/V）, glycerin4.5%（V/V）, and trehalose0.1%（W/V）. The complex protective agents could improve thermal stability of β-glucanasebetween50℃and90℃, and also reduced the rate of heat degradation2.8times,significantly improved the storage stability.