CAJ | 학술논문

基于来源于Bacillus cereus WQ92的耐有机溶剂蛋白酶WQ的液相色谱双质谱（ LC MS MS）分析结果，设计引物，克隆耐有机溶剂蛋白酶WQ的基因，测序分析表明该蛋白酶的开放阅读框（ ORF）大小为1701 bp，编码566个氨基酸，其中含有信号肽（28个氨基酸）、前肽（220个氨基酸）及成熟肽序列（含954 bp编码318个氨基酸），相对分子质量约为3?7×104。将不带自身信号肽的蛋白酶基因aprWQ插入穿梭质粒pMA5中，构建了表达载体pMA5／aprWQ。该表达载体导入枯草芽胞杆菌WB600中获得阳性重组子WB600 pMA5／aprWQ，通过优化培养基成分以及培养条件，重组子发酵产蛋白酶体积酶活达到17400 U／mL，约为高产野生菌产酶量的5倍。重组蛋白酶在多种有机溶剂（体积分数为50％）中表现出了良好的耐受性，验证了重组菌表达的蛋白酶与来源于B?cereus WQ92的耐有机溶剂蛋白酶性质一致，该耐有机溶剂蛋白酶的高效表达为进一步发挥其高效生物催化作用等实际应用奠定了基础。
기우래원우Bacillus cereus WQ92적내유궤용제단백매WQ적액상색보쌍질보（ LC MS MS）분석결과，설계인물，극륭내유궤용제단백매WQ적기인，측서분석표명해단백매적개방열독광（ ORF）대소위1701 bp，편마566개안기산，기중함유신호태（28개안기산）、전태（220개안기산）급성숙태서렬（함954 bp편마318개안기산），상대분자질량약위3?7×104。장불대자신신호태적단백매기인aprWQ삽입천사질립pMA5중，구건료표체재체pMA5／aprWQ。해표체재체도입고초아포간균WB600중획득양성중조자WB600 pMA5／aprWQ，통과우화배양기성분이급배양조건，중조자발효산단백매체적매활체도17400 U／mL，약위고산야생균산매량적5배。중조단백매재다충유궤용제（체적분수위50％）중표현출료량호적내수성，험증료중조균표체적단백매여래원우B?cereus WQ92적내유궤용제단백매성질일치，해내유궤용제단백매적고효표체위진일보발휘기고효생물최화작용등실제응용전정료기출。
Based on the liquid chromatography?mass?mass spectrometry ( LC?MS?MS) analysis of trypsin?digested protein fragments of the solvent?stable protease WQ from a solvent?stable strain Bacillus cereus WQ9?2,solvent?stable protease gene was successfully cloned. The gene contains an open reading frame of 1 701 bp,encoding a pre?pro?protein enzyme of 566 amino acids(28?aa pre?signal peptide,220?aa pro?peptide and 318?aa mature protein with the molecular mass of 3?7×104). The expression plasmid pMA5/aprWQ was constructed by inserting the aprWQ gene into the vector pMA5 without native signal peptide sequence. The maximum concentration of the recombinant protease was 17 400 U/mL,5 fold higher than the natural production level. Molecular weight, tolerance in organic solvent of recombinant solvent?stable protease was identical to the native protease. The findings provides the basis for further expansion of the catalytic applications of organic solvent?stable protease.