You might try something like the following.

use strict;
use warnings;
use DB_File;

my $hashfile = "hash.$$";
tie my %hash1, "DB_File", $hashfile, O_RDWR|O_CREAT, 0666, $DB_HASH
    or die "cannot open file $hashfile: $!";


open(my $fh1, "<", "file1.txt") or die "file1.txt: $!";
foreach (<$fh1>) {
    chomp;
    my @parts = split(/\s+/);
    $hash1{"$parts[1]#$parts[2]"} = $parts[4];
}
close($fh1);


open(my $fh2, "<", "file2.txt") or die "file2.txt: $!";
foreach (<$fh2>) {
    my @parts = split(/[\s>]+/);
    my $value = $hash1{"$parts[0]#$parts[1]"};
    if( defined($value) and grep { $_ eq $parts[2] } split(/\//, $valu
+e)) {
        print "$_";
    }
}
close($fh2);


untie %hash1;
unlink($hashfile);
[download]

Using a tied hash will allow you to process larger data sets - where an in-memory hash would exceed available memory. You may not need this but a 500MB file will result in quite a large hash. Performance will be better if you can use an in-memory hash (i.e. if you don't use the tied hash).

Only one hash is built. There is no benefit building a hash from the second file as you never do lookup in that hash.

Some of your matching criteria weren't clear to me. Your descriptions and code seemed to differ and I wasn't sure what "single character" means with respect to a value like "A/TTA/AT". You may have to make some changes to the matching criteria in the second loop.