Characterization Of Thermostable β-Glucanase Produced By Bacillus Licheniformis ZJU0107 And Its Application To Beer Production

glucanase is an important enzyme that may largely reduce the negative effects of cereal p-glucan on the processes of beer and feed industries. In this study, an isolate of Bacillus, lichniformis ZJU0107 from soil was found producing a thermostable |5-glucanase at high yield. Production of this enzyme was investigated by optimizing components of medium and conditions of fermentation. The fi-glucanase of B. lichniformis ZJU0107 was purified and characterized for its properties and kinetics. The partitioning behavior of the |5-glucanase in different aqueous two-phase systems was investigated. The application of its preparation to mashing process in beer production was evaluated in parallel to a commercial product. The major results are summarized as follows.Bacterial isolate and optimized conditions for high production of p*-ghicanase. A bacterial isolate producing the (3-glucanase at high level was selected from numerous soil isolates and identified as Bacillus lichniformis. The p-glucanase produced by this isolate was most active at 60癈. The isolate was then mutated by exposure to ultraviolet, followed by chemical pressure of diethyl sulfate. The selected mutant B. lichniformis ZJU0107 was found producing p-glucanase 2.44 times more than the original isolate. Inclusion of barley-sourced (3-glucan into medium further enhanced the p-glucanase production. An optimized medium for the production of the enzyme by the mutant was composed of bran 14.1 g/L, fish meal 8.0 g/L, KNO3 3.4 g/L, and MgSO4-7H2O 1.1 g/L. The optimal conditions for fermentation were determined as 36癈, initial pH 6.0, and 3.3% (v/v) seed culture containing 108 cells/mL. A 52-h incubation of the mutant in the liquid medium under the optimized conditions resulted in a fJ-glucanase activity level of 115.1 U/mL.The mutant produced not only the p-glucanase but also xylanase and neutral protease at high levels. The three enzymes were produced in association with cell growth; their activities increased with the increasing biomass during the log-growth stage and were highly expressed at the stationary phase.Purification and characterization of the B. lichniformis ZJU0107 fi-glucanase. The 6-glucanase produced by B. lichniformis ZJU0107 was purified by means of SephadexG-100 and DEAE-Sephadex A-50 chromatography, and then was characterized for its properties and kinetics. Its molecular mass was determined as 31 kDa in the SDS-PAGE analysis and its basic isoelectric point as 9.1. The enzyme was able to specifically hydrolyze (l-3,l-4)-B- bonds in barley-sourced B-glucans and lichenins, but did not hydrolyze CM-cellulose and laminarin. This indicates that the enzyme can be classified to 6-1-3,1-4-glucanase (code: E.C.3.2.1.73).The purified B-glucanase had maximal activity at pH 5.5 and its relative activity exceeded 80% after maintenance at pH 5.0-10.0 for 5 h. Its optimal reaction temperature ranged from 55 to 65癈 with maximal activity at 60癈. Its activity could be suppressed at the presence of Fe3+and A13+ but conspicuously stimulated by Fe2+, Co2+ or Ca2*, especially by Co2+. However, other metal ions including K+, Na+, Mn2+ or Mg2* had little effects on its activity. When barley-sauced 6-glucan was used as substrate, parameters Km and Vaax for the B-glucanase kinetics were estimated as 3.855 mg/mL and 0.1248 mg/mL/min, resulting in the kinetic model Vp -0.1248C5 7(3.855+ C,). When lichenan was used as substrate,the Km and Kmaxwere estimated as 6.351 mg/mL and 0.1951 mg/mL/min, generating the kinetic model Vp - 0.1951C, /(6.351 + C,).Harvest of enzymes from liquid culture. The partition behavior of 6-glucanase, xylanase and neutral protease produced in the liquid culture of B. lichniformis ZJU0107 or in its supernatant was investigated using gradient PEG-phosphate systems. The effects of PEG molecular weight and concentration, salt concentration, pH and NaCl concentration on the partition and extraction of the three enzymes in the two-phase systems were examined. As a result, the PEG molecular weight and concentration were found largely affe...