Conversion of Lactococcus lactis cell envelope proteinase specificity by partial allele exchange

Abstract

Aims: To determine whether conversion of lactocepin substrate binding regions by gene replacement can alter lactocepin specificity in Lactococcus lactis starter bacteria without affecting other important strain properties. Methods and Results: We utilized two‐step gene replacement to convert substrate‐binding determinants in the L. lactis prtP genes encoding group h (bitter) lactocepin in two industrial strains into the corresponding group b (nonbitter) variant. Analysis of lactocepin activity toward αs1‐casein (f 1–23) by reversed‐phase high‐pressure liquid chromatography demonstrated enzyme specificity among isogenic derivatives had been altered in a manner that was consistent with predicted amino acid substitutions in substrate binding regions. Milk acidification properties of some mutants were not statistically different (P > 0·05) from wild‐type parent strains, and strain propensity for autolysis was also not significantly (P > 0·05) changed. Conclusions: Conversion of lactocepin substrate binding regions by allele exchange can effectively alter lactocepin specificity in industrial strains of L. lactis without significantly affecting other important strain properties. Significance and Impact of the Study: Methodology outlined in this study can be used to alter lactocepin specificity in commercial starter cultures with a propensity for bitter flavour defect, and prtP derivatives developed by this approach should be suitable for commercial application.

Invited review: Starter lactic acid bacteria survival in cheese: New perspectives on cheese microbiology

“…Groups a (example strain SK11) and b (strain AM2, S2) CEP proteins are associated with nonbitter strains, whereas groups g (strain HP) and h (strain S3) have been associated with bitter peptide production. Broadbent et al (2006) showed that autolysis was unaffected by allelic exchange of catalytic sites among groups b and h, but detection of LDH was done on 24-h LM17 culture supernatants, which may have mitigated any differences among the strains. Viability of bitter strains in Cheddar cheese curd was higher on d 1 of ripening than that of nonbitter starter strains, whereas the latter subsequently showed greater loss of viability during extended ripening (Martley and Lawrence, 1972;Wilkinson et al, 1994).…”

Genetic diversity in proteolytic enzymes and amino acid metabolism among Lactobacillus helveticus strains

“…Research with the highly related (98% AA sequence identity) CEP alleles found in Lc. lactis has shown that these enzymes may be differentiated by their relative affinity and specificity for individual CN (Kunji et al, 1996), and that these differences can impart significantly different effects on the peptide profiles and sensory quality of cheese (Broadbent et al, 2002(Broadbent et al, , 2006Pillidge et al, 2003). Enzymes from Lb.…”

Improved growth of bifidobacteria by cocultivation with Lactococcus lactis subspecies lactis

“…Extraction of DNA from lactococcal strains was performed using the DNeasy Blood & Tissue Kit according to the manufacturer's instructions (Qiagen Sciences, Gaithersburg, MD). Polymerase chain reaction detection of the prtP gene was performed as described previously (Broadbent et al, 2006). Briefly, the PCR mixture was performed using TaKaRa Ex Taq (Takara Bio Inc., Shiga, Japan) according to the manufacturer's instructions.…”