(浙江工业大学生物工程学院 杭州 310014)
(College of Biotechnology and Bioengineering， Zhejiang University of Technology， Hangzhou 310014)
天然抗氧化功能的活性多肽和食品酶具有绿色、安全及可靠的特点，广受欢迎。己糖氧化酶是一种底物谱广且具有除氧、酸化、提高蛋白交联等功能的氧化酶，可作为一种新型绿色多功能的食品添加剂。为了提高己糖氧化酶在毕赤酵母中的过量表达，系统比较不同基因型的宿主菌株、表达方式以及多拷贝数转化子等因素对己糖氧化酶异源表达的影响。应用新建立的3步筛选法获得不同基因型的转化子并比较其发酵酶活，结果表明，甲醇代谢缓慢的Muts型宿主有利于己糖氧化酶的表达；而甲醇诱导表达方式优于组成型表达方式；较高的己糖氧化酶基因拷贝数表达相对更高的酶活。然而，优化以上表达因素，己糖氧化酶表达水平仍不高，最高摇瓶发酵酶活为4.92×10-2 U/mL。重组酶的纯化研究表明存在3种蛋白，分子质量分别为62，40 ku和29 ku，即己糖氧化酶的原酶和经蛋白酶切割而成的2个二聚体亚基，其中原酶的活性较低且占比最高，可能是表达活性偏低的原因之一。酶学特性研究表明，重组酶的最适条件为50~60 ℃、pH 4.0，且常温条件(20 ℃)下可维持50%的活性。低温条件(20~60 ℃)和酸性条件(pH 3.0~6.0)，己糖氧化酶酶活较为稳定，说明其是一种耐酸性较好的抗氧化酶，适用于酸性食品的保鲜和抗氧化应用。
Active peptides or food enzymes with natural antioxidant function are green， safe and reliable， and have attracted wide interest as green and multifunctional food additives. Hexose oxidase (HOX)， a D-hexose:O2 1-oxidoreductase， and has the functions of deoxygenation， acidification and improving protein crosslinking. In order to optimize the expression strategies of hexose oxidase in Pichia pastoris， different genotype host strains， different gene expression modes and different gene copy number in transformants were systematically compared. The transformants with different genotypes were obtained by the newly established three-step screening method， and their enzyme activities by fermentation were compared. The results showed that Muts host with slow methanol metabolism generated higher HOX activity； inducible expression by methanol was better than constitutive expression by GAP promoter； higher gene copy number of HOX gene generated higher enzyme activity. Although these factors related to gene expression in P. pastoris had been optimized， the HOX activity was still in a low level， and the maximum activity produced by KCX56 strain was 4.92×10-2 U/mL， which contained six gene copy number. The purification of recombinant HOX showed that there were three recombinant proteins with the molecular weight of 62， 40 ku and 29 ku， respectively. They should be protoenzyme of hexose oxidase and two subunits formed by proteolytic cleavage. Among them， the proportion of protoenzyme with low activity was much higher than other two， which may be one of the reasons for the low fermentation activity by these recombinant strains. The study of enzymatic characteristics showed that the optimum conditions for the recombinant HOX were 50-60 ℃ and pH 4.0， and it can maintain 50% activity at room temperature (20 ℃). Under low temperature (20-60 ℃) and acidic conditions (pH 3.0-6.0)， HOX activity can be stably maintained， so it is an antioxidant enzyme with good acid resistance， meaning that HOX is suitable for the preservation and antioxidant application for acidic foods.