关键词: 甘露聚糖酶, 果汁澄清, 异源表达, 食品酶, 嗜热酶

Abstract: A β-mannanase gene, nsMan5B, was cloned from the thermophilic fungus Neosartorya sp. HBFH9 and successfully expressed in Pichia pastoris. Sequence analysis indicated that the complete DNA sequence of nsMan5B was 1 491 bp in length (containing 2 Introns). The cDNA sequence of nsMan5B was 1 371 bp in length, which could encode a protein of 456 amino acids with a molecular mass of 49 kDa. Structural analysis indicated the presence of a putative signal peptide, a carbohydrate-binding module of family 1 (CBM 1), a linker region and a catalytic domain of GH5 in NsMan5B. The recombinant NsMan5B was optimally active at pH 4.0 and 60 ℃, and had good stability over the wide range of pH 2.0–10.0 and at 50 ℃ or below. The effects of metal ions and chemical reagents on its enzymatic activity were different. The enzymatic activity was increased by 271%, 123% and 100% in the presence of K+, Mg2+ and Cu2+, respectively. With increasing carbon chain length and concentration of alcohol, the inhibitory effect on NsMan5B activity was markedly enhanced. Its enzymatic activity was reduced by 86% by n-butanol (2.5%, V/V). It was observed that NsMan5B was effective in the clarification of orange juice as well as persimmon, apple, peach and grape juices, whose clarity was increased by 31.8%, 7%, 4% and 4%, respectively. Consequently, it was concluded that the NsMan5B could be successfully used in the fruit juice industry.

Key words: mannanase, juice clarification, expression, food enzymes, thermophilic enzyme