雙叉桿菌β-半乳糖苷酶之生產、特性及其於半乳寡醣合成之應用

Production and characterization of β-galactosidase from bifidobacteria and its application in galactooligosaccharides synthesis

徐志安(Chih-An Hsu)

指導教授 ： 周正俊

國立臺灣大學/生物資源暨農學院/食品科技研究所/博士(2006年)

https://doi.org/10.6342/NTU.2006.01400

摘要

部分微生物所產生之β-半乳糖苷酶(β-galactosidase)除具水解乳糖之能力外，亦可表現出轉半乳糖活性(transgalactosylation)以產生具益菌助生質(prebiotics)功能之半乳寡醣(galactooligosaccharides)。本研究乃利用雙叉桿菌生產具轉半乳糖活性之β-半乳糖苷酶，並進一步將此酵素純化以探討β-半乳糖苷酶之特性；爾後，利用此酵素進行轉半乳糖反應以合成半乳寡醣。
在三角錐瓶系統下，於所測試之雙叉桿菌菌株中，以B. longum BCRC 15708能產生最高之β-半乳糖苷酶活性及比活性。當以B. longum BCRC 15708做為試驗菌株時發現，生產β-半乳糖苷酶之最適碳源及氮源分別為lactose及yeast extract；並於含4% lactose、3.5% yeast extract、0.3% K2HPO4、0.1% KH2PO4、0.05% MgSO4•7H2O及0.03% L-cysteine之培養液組成中，與在培養液初pH值及培養溫度分別為6.5及37℃下，當培養時間為16小時時，可得最高之β-半乳糖苷酶活性，為18.6 U/ml。
B. longum BCRC 15708之β-半乳糖苷酶經Q Fast Flow陰離子交換樹酯層析及Superose 6 HR膠體過濾層析純化後，可得純化倍數15.7倍及酵素比活性168.6 U/mg之β-半乳糖苷酶，並以Native-PAGE分析可得其分子量為357 kDa。當以o-nitrophenyl-β-D-galactopyranoside (ONPG)做為反應基質時，酵素反應之最適作用溫度及pH值分別為50℃及7.0，最大反應速率(Vmax)及反應速率常數(Km)則分別為70.67 U/mg及0.85 mM。鈉離子及鉀離子可促進酵素之活性，約可提高達10倍；然而，1 mM之鐵離子及二價陽離子(亞鐵離子、鈷離子、銅離子、鈣離子、鋅離子、錳離子及鎂離子)則會造成β-半乳糖苷酶活性被嚴重抑制之結果。半乳糖、乳糖及果糖等亦均會抑制β-半乳糖苷酶活性。
利用批式5公升發酵槽培養B. longum BCRC 15708時發現，接種量、培養溫度、培養液pH值及攪拌速率等均會影響β-半乳糖苷酶之生產。當接種量、培養溫度、培養液pH值及攪拌速率分別為20% (v/v)、37℃、6.5及100 rpm，且初始乳糖及酵母抽出物添加濃度分別為4%及3.5%時，經10小時發酵培養後，可得最高β-半乳糖苷酶活性及轉半乳糖活性，分別為36.7 U/ml及0.49 U/ml。
當以B. longum BCRC 15708之β-半乳糖苷酶行轉半乳反應時，可產生二種半乳寡醣，分別為3糖及4糖，其中又以3糖為主要之半乳寡醣。於乳糖濃度為40%，反應液pH值及反應溫度分別為6.8及45℃下，當乳糖之轉化率為57.8%時，可得最大之半乳寡醣產率(yield)及生產力(productivity)，分別為30.1% (w/w)及13.9 g/l•h；又當葡萄糖及半乳糖等單醣之添加濃度為15%以上時，則會對於β-半乳糖苷酶之轉半乳糖反應造成抑制的效果，達17.3-35.8%。

關鍵字

雙叉桿菌 ； β-半乳糖苷酶半乳寡醣 ； 轉半乳糖反應

並列摘要

β-Galactosidase produced by some microorganisms, in addition to catalyze the hydrolysis of lactose, exhibits a transgalactosylation activity (TGase activity) which can catalyze the formation of galactooligosaccharides (GOS), a prebiotics, from lactose. In this study, experiments were conducted to investigate the production of β-galactosidase which processes transgalactosylation activity from Bifidobacterium. In addition, purification and characterization of β-galactosidase from B. longum BCRC 15708 as well as the synthesis of GOS with this enzyme were performed.
β-Galactosidase production by Bifidobacterium strains, including B. longum BCRC 14634, 15708 as well as B6, B. breve BCRC 11846, B. bifidum BCRC 14615, B. adolescentis BCRC 14608, and B. infantis BCRC 14633, was first carried out in flask, with B. longum BCRC 15708 showing the highest production of β-galactosidase with the highest specific activity. Further study with B. longum BCRC 15708, it was first found that lactose and yeast extract, respectively, were the best carbon source and nitrogen source for β-galactosidase production. After 16 hrs cultivation, a maximum β-galactosidase activity of ca 18.6 U/ml was obtained in medium containing 4% lactose, 3.5% yeast extract, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO4•7H2O, 0.03% L-cysteine, and with the initial pH value and temperature controlled at 6.5 and 37℃, respectively.
β-Galactosidase from B. longum BCRC 15708 was purified by procedures including Q Fast-Flow chromatography and Superose 6 HR gel chromatography. These steps resulted in a purification of 15.7-fold, a yield of 29.3% and a specific activity of 168.6 U mg-1 protein. The molecular weight was 357 kDa as determined from Native-PAGE. Using o-nitrophenyl-β-D-galactopyranoside (ONPG) as substrate, the pH and temperature optimum of the purified β-galactosidase were 7.0 and 50℃, respectively. The Km and Vmax were 0.85 mM and 70.67 U/mg, respectively. Monovalent cations (Na+ and K+) stimulated the enzyme up to 10-fold, whereas Fe3+ and bivalent cations (Fe2+, Co2+, Cu2+, Ca2+, Zn2+, Mn2+, and Mg2+) in the concentration of 1 mM inhibit β-galactosidase activity. Furthermore, galactose, lactose, and fructose would also inhibit the enzyme activity.
B. longum BCRC 15708 grown in a jar fermenter showed that inoculum size, cultivation temperature, the pH of medium and agitation speed all would affect β-galactosidase production. A maximum β-galactosidase activity of 36.7 U/ml and a maximum transgalactosylation activity of 0.49 U/ml were achieved after 10 hr of fermentation with 20% inoculum of the test organism into a medium containing 4% lactose and 3.5% yeast extract, and with the pH of medium, cultivation temperature and agitation speed controlled at 6.5, 37℃ and 100 rpm, respectively.
Two types of GOS, tri- and tetrasaccharides, were formed by transgalactosylation catalyzed by β-galactosidase from B. longum BCRC 15708, while trisaccharides were the major type of GOS formed. A maximum yield of 30.1% (w/w) GOS could be achieved from 40% lactose solution at 45℃ and pH 6.8 when the lactose conversion was 57.8%. The corresponding productivity of GOS was 13.9 g/l•h. Moreover, when more than 15% of galactose or glucose was added to reaction mixture containing 40% lactose, transgalactosylation reaction was markedly inhibited by 17.3-35.8%.

並列關鍵字

Bifidobacteria ； β-galactosidase ； galactooligosaccharides ； transgalactosylation

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