Novel high-performance metagenome β-galactosidases for lactose hydrolysis in the dairy industry

Abstract

The industrially utilised β-galactosidases from Kluyveromyces spp. and Aspergillus spp. feature undesirable kinetic properties in praxis, such as an unsatisfactory lactose affinity (KM) and product inhibition (KI) by galactose. In this study, a metagenome library of about 1.3 million clones was investigated with a three-step activity-based screening strategy in order to find new β-galactosidases with more favourable kinetic properties. Six novel metagenome β-galactosidases (M1-M6) were found with an improved lactose hydrolysis performance in original milk when directly compared to the commercial β-galactosidase from Kluyveromyces lactis (GODO-YNL2). The best metagenome candidate, called "M1", was recombinantly produced in Escherichia coli BL21(DE3) in a bioreactor (volume 35 L), resulting in a total β-galactosidase M1 activity of about 1100 μkatoNPGal,37 °C L(-1). Since milk is a sensitive and complex medium, it has to be processed at 5-10 °C in the dairy industry. Therefore, the β-galactosidase M1 was tested at 8 °C in milk and possessed a good stability (t1/2=21.8 d), a desirably low apparent KM,lactose,8 °C value of 3.8±0.7 mM and a high apparent KI,galactose,8 °C value of 196.6±55.5 mM. A lactose hydrolysis process (milk, 40 nkatlactose mLmilk,8 °C(-1)) was conducted at a scale of 0.5L to compare the performance of M1 with the commercial β-galactosidase from K. lactis (GODO-YNL2). Lactose was completely (>99.99%) hydrolysed by M1 and to 99.6% (w/v) by K. lactis β-galactosidase after 25 h process time. Thus, M1 was able to achieve the limit of <100 mg lactose per litre milk, which is recommended for dairy products labelled as "lactose-free".

Functional metagenomics reveals novel β-galactosidases not predictable from gene sequences

“…meliloti can hydrolyze lactose but not X-Gal, suggestiong that X-Gal was not a good fit with the active sites of those enzymes. The same property of other metagenomic β-galacctosidases has been reported [28]. Here we have highlighted the application of functional metagenomics for mining novel enzymes from soil microbial communities.…”

“…The associated transgalactosylation activity of β-galactosidases can be used to synthesize galactosylated products [24]. Functional screening of metagenomic libraries resulted in discovery of a GH43 enzyme acting on multiple substrates including lactose [25], cold-adapted or thermostable β-galactosidases [26–28], GH1/GH2 [29–33], two novel β-galactosidases without any similarity to known GHs [7], glycosyltransferase family 4 and β-glycosidase with β-galactosidase activity respectively [34, 35]. …”

Ciência e Tecnologia de Leite e Produtos Lácteos Sem Lactose

“…Novas β-galactosidases bem como tecnologias inovadoras ligadas à produção dessa enzima vêm sendo descobertas e estudadas pelos pesquisadores (DUAN et al, 2017;JOSE et al, 2017;LIU et al, 2017;YOU, Chang, et al, 2017;YOU, Zhang, et al, 2017). Erich et al (2015) realizaram uma triagem de 1,3 milhões de clones de metagenoma para descobrir novas β-galactosidases. Seis β-galactosidases de metagenoma foram descobertas e apresentaram melhor desempenho de hidrólise da lactose no leite quando comparado com a β-galactosidase comercial de Kluyveromyces lactis (nome comercial GODO-YNL2, produzida pela Shusei Co, Ltd., Tóquio, Japão).…”

A highly inducible β-galactosidase from Enterobacter sp.

“…It can hydrolyze lactose, mainly being used in food technology for lactose hydrolysis in milk and milk by-products. 6 It can also go through a process called transgalactosylation, taking part in the production of a variety of trans-galactosylation products or prebiotic galacto-oligosaccharides (GOS). It was recently found that these prebiotic products stimulate the growth and establishment of Bifidobacteria in the intestine of human and overcome potentially harmful bacteria, such as species belonging to Clostridia and Bacteriodes in the gut and are now considered as prebiotic.…”