Agriculture is an essential industry to New Zealand. Despite its importance, 52% of the total carbon dioxide equivalent (CO2-e) emissions for New Zealand came from the agriculture sector in 2020; of which 49% of those agricultural emissions originated from enteric fermentation. Methanogens in the rumen of animals produce methane as a by-product of enteric fermentation and are a target for methane mitigation. There are currently very few methane mitigation strategies for grazing livestock tailored towards inhibiting methanogen strains. The dominant methanogens in the rumen of New Zealand animals have a cell wall made of pseudomurein and cannot be lysed by general cell wall antibiotics. Previous studies have identified proteins originating in viruses and phages able to specifically lyse, or otherwise inactivate methanogen cells, called pseudomurein endoisopeptidases. The CRISPR-Cas adaptive immunity of methanogens allows for the study of key virus-host interactions to determine viral mechanisms for methanogen inhibition. Here, two independent approaches were used to identify novel pseudomurein endoisopeptidase (Pei) enzymes based on their similarity to those previously characterised, PeiP, PeiW, and PeiR. The first approach used a homology search of the NCBI protein database and metagenomes from rumen microbiome data; both filtered for methanogen prevalence in sheep. The second approach identified for the first time using CRISPRTarget, methanogen specific viruses in the IMG/VR and Genbank phage databases, whose genomes were then annotated for potential Pei proteins. A total of 21 novel Pei proteins were identified that contained the same domains as the known Pei proteins, necessary for the lysis of methanogen cells. Of those, 5 were chosen for experimental characterisation based on their homology to the PeiR protein. The 3 proteins PeiG2*, PeiTh2*, and PeiF3* were unable to cleave the L-Ala-pNA synthetic peptide designed to mimic the peptide bonds in pseudomurein. However, all three proteins were able to lyse a cell suspension of Methanobrevibacter sp. AbM4; two of which, PeiTh2* and PeiF3*, were significantly more effective than the positive control PeiR. Mutations in conserved amino acids in the PMBR and active site domains of PeiF3* revealed that the highly conserved prolines in the PMBR domain and the conserved active site residues in the C39 peptidase domain, convey binding and lytic activity, respectively, of the Pei protein. This was shown by the repressed activity of the two mutant proteins, PeiF3_P and PeiF3_A, on a cell suspension of Methanobrevibacter sp. AbM4 when compared with the native PeiF3* protein. The findings of this study validate a novel search method for pseudomurein endoisopeptidase proteins using CRISPRTarget and classify 3 new active Pei proteins for further development with regards to methane mitigation.