Malaysia has expelled North Korea’s ambassador, giving him 48 hours to leave the country in a major break in diplomatic relations over the assassination of the half-brother of the North Korean leader.

Kim Jong-nam was poisoned on 13 February with deadly nerve agent VX. North Korea has repeatedly disparaged the murder investigation, accusing Malaysia of conniving with its enemies.

The ambassador was declared persona non grata after Malaysia demanded but did not receive an apology for Pyongyang’s attacks on the investigation, Malaysia’s foreign minister, Anifah Haji Aman, said.

“Malaysia will react strongly against any insults made against it or any attempt to tarnish its reputation,” he said in a statement released late on Saturday.

Ambassador Kang Chol failed to present himself at the ministry when summoned and is expected to leave Malaysia within 48 hours, the statement added. The deadline is 6pm on Monday.

South Korea has blamed the North for the murder, citing what they say was a standing order from leader Kim Jong-un to kill his exiled half-brother who may have been seen as a rival.

The foreign ministry said the expulsion was “part of the process by the Malaysian government to review its relations” with North Korea, which before Kim’s assassination were unusually cosy.

“North Korea must learn to respect other countries,” Malaysia’s deputy prime minister, Ahmad Zahid Hamidi, said.

The expulsion shows “we are serious about solving this problem and we do not want it to be manipulated”, he added.

On Sunday evening, a senior government official who did not want to be named said Kang was still in the country and was expected to leave on a flight to Beijing on Monday.

The diplomatic spat erupted last month when Malaysian police rejected North Korean diplomats’ demands to hand over Kim’s body. Kang then claimed the investigation was politically motivated and said Kuala Lumpur was conspiring with “hostile forces”.

Malaysia summoned Kang for a dressing-down, with the prime minister, Najib Razak, saying the ambassador’s statement was “diplomatically rude”.

Malaysia issued a deadline of 28 February for an apology, but “no such apology has been made, neither has there been any indication that one is forthcoming”.

Malaysia has also recalled its envoy to Pyongyang and cancelled a visa-free travel deal with North Korea.

Police are seeking seven North Korean suspects but on Friday released the only North Korean arrested because of lack of evidence.

After Ri Jong-chol was deported, he claimed police offered him a comfortable life in Malaysia for a false confession, saying the investigation was “a conspiracy to impair the dignity of the Republic [North Korea]”.

Two women – one Vietnamese and one Indonesian – have been charged with murdering Kim Jong-nam, with airport CCTV footage showing them approaching the 45-year-old and apparently smearing his face with a cloth.

Police say he had a seizure and died less than 20 minutes later. Swabs of the dead man’s face revealed traces of VX.

North Korea had few friends even before the assassination, but the fallout from the killing looks set to further isolate the nuclear-armed state.

Malaysia formally established diplomatic relations with North Korea in 1973 and opened an embassy in Pyongyang in 2003. It has provided a conduit between Pyongyang and the wider world in recent years, with Kuala Lumpur serving as a discreet meeting place for talks between the regime and the US.

Up to 1,000 North Koreans work in Malaysia and their remittances are a valuable source of foreign currency for the isolated regime.

Last week, Malaysia’s trade minister, Mustapa Mohamed, said the spat would have no impact on Kuala Lumpur as trade with the reclusive country is “insignificant”.