工业微生物
工業微生物
공업미생물
INDUSTRIAL MICROBIOLOGY
2015年
1期
36-42
,共7页
产酸克雷伯氏菌%普鲁兰酶%异源表达%蛋白纯化%酶学性质
產痠剋雷伯氏菌%普魯蘭酶%異源錶達%蛋白純化%酶學性質
산산극뢰백씨균%보로란매%이원표체%단백순화%매학성질
Klebsiella oxytoca%pullulanase%heterologous expression%enzymatic properties
普鲁兰酶（EC 3．2．1．41）是一类淀粉脱支酶，能够特异性水解淀粉中的α-1，6-糖苷键，从而提高淀粉的利用率，在以淀粉为原料的食品、纺织、生物燃料和洗涤剂等行业中具有重要的应用价值。本研究以产酸克雷伯氏菌Klebsiella oxytoca M5 al基因组DNA为模板，将PCR扩增得到的普鲁兰酶基因pulA克隆至表达载体pET28 a （＋），构建好的重组质粒转化大肠杆菌Escherichia coli BL21（DE3），在培养基中添加0．5 mmol／L异丙基硫代半乳糖苷（IPTG）的条件下对该酶基因进行诱导表达，经镍柱纯化获得重组普鲁兰酶用于酶学性质研究。SDS-PAGE及Western Blot检测显示普鲁兰酶基因pulA在上述大肠杆菌宿主中成功获得了表达。该重组酶最适反应pH5 ．5 ，最适温度60℃。金属离子对酶活性有一定影响。Mn2＋对酶活促进作用显著；Fe3＋、Mg2＋、Fe2＋对酶活只有微弱的促进作用，而Cu2＋对酶活造成强烈抑制。来源于Klebsiella oxytoca M5al的普鲁兰酶最适催化条件符合工业生产中淀粉糖化工艺的要求，具有应用于淀粉工业的潜力。
普魯蘭酶（EC 3．2．1．41）是一類澱粉脫支酶，能夠特異性水解澱粉中的α-1，6-糖苷鍵，從而提高澱粉的利用率，在以澱粉為原料的食品、紡織、生物燃料和洗滌劑等行業中具有重要的應用價值。本研究以產痠剋雷伯氏菌Klebsiella oxytoca M5 al基因組DNA為模闆，將PCR擴增得到的普魯蘭酶基因pulA剋隆至錶達載體pET28 a （＋），構建好的重組質粒轉化大腸桿菌Escherichia coli BL21（DE3），在培養基中添加0．5 mmol／L異丙基硫代半乳糖苷（IPTG）的條件下對該酶基因進行誘導錶達，經鎳柱純化穫得重組普魯蘭酶用于酶學性質研究。SDS-PAGE及Western Blot檢測顯示普魯蘭酶基因pulA在上述大腸桿菌宿主中成功穫得瞭錶達。該重組酶最適反應pH5 ．5 ，最適溫度60℃。金屬離子對酶活性有一定影響。Mn2＋對酶活促進作用顯著；Fe3＋、Mg2＋、Fe2＋對酶活隻有微弱的促進作用，而Cu2＋對酶活造成彊烈抑製。來源于Klebsiella oxytoca M5al的普魯蘭酶最適催化條件符閤工業生產中澱粉糖化工藝的要求，具有應用于澱粉工業的潛力。
보로란매（EC 3．2．1．41）시일류정분탈지매，능구특이성수해정분중적α-1，6-당감건，종이제고정분적이용솔，재이정분위원료적식품、방직、생물연료화세조제등행업중구유중요적응용개치。본연구이산산극뢰백씨균Klebsiella oxytoca M5 al기인조DNA위모판，장PCR확증득도적보로란매기인pulA극륭지표체재체pET28 a （＋），구건호적중조질립전화대장간균Escherichia coli BL21（DE3），재배양기중첨가0．5 mmol／L이병기류대반유당감（IPTG）적조건하대해매기인진행유도표체，경얼주순화획득중조보로란매용우매학성질연구。SDS-PAGE급Western Blot검측현시보로란매기인pulA재상술대장간균숙주중성공획득료표체。해중조매최괄반응pH5 ．5 ，최괄온도60℃。금속리자대매활성유일정영향。Mn2＋대매활촉진작용현저；Fe3＋、Mg2＋、Fe2＋대매활지유미약적촉진작용，이Cu2＋대매활조성강렬억제。래원우Klebsiella oxytoca M5al적보로란매최괄최화조건부합공업생산중정분당화공예적요구，구유응용우정분공업적잠력。
Pullulanase(EC3. 2. 1. 41),one of debranching enzymes,can specifically hydrolyze α-1,6-glucosidic bonds in starch and greatly promote starch utilization. Pullulanase is widely used in food,textile,biofuel and detergent industry. In present study,pullulanase gene pulA was amplified by PCR using genomic DNA of Klebsiella oxytoca M5al as template, then cloned into vector pET28a(+)and expressed in Escherichia coli BL21 (DE3)induced by 0. 5 mmol/L IPTG. The expression of recombinant pullulanase was confirmed by SDS-PAGE and western blot. The enzyme was purified by Ni-Agarose resin and its enzymatic properties were determined. The optimum pH and temperature of recombinant enzyme were 5. 5 and 60 ° C respectively. The activity of recombinant enzyme was significantly increased while adding Mn2+,but slightly enhanced by adding Fe3+,Mg2+ and Fe2+. The enzyme activity was greatly inhibited in the presence of Cu2+. The optimal catalytic conditions of pullulanase from Klebsiella oxytoca M5 al met the requirement of saccharification process,showing its application potential in starch industry.
